Methods of selecting internalizing antibodies

ABSTRACT

This invention provides methods of selecting antibodies that are internalized into target cells. The methods generally involve contacting target cells with one or more members of an antibody phage display library. The members of the phage display library are also contacted with cells of a subtractive cell line. The target cells are then washed to remove the subtractive cell line cells and members of the phage display library that are non-specifically bound or weakly bound to the target cells. The target cells are cultured under conditions where members of the phage display library can be internalized if bound to an internalizing marker and internalized members of the phage display library are then identified.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/855,755 filed May26, 2004, which application is a divisional of and claims benefit ofU.S. Ser. No. 09/249,529 filed Feb. 12, 1999, which claims benefit under35 U.S.C. §119(e) of provisional application U.S. Ser. No. 60/082,953,filed on Apr. 24, 1998, which application are herein incorporated byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported, in part, by Department of Defense GrantsDAMD17-96-1-6244 and DAMD17-94-4433. The government of the United Statesof America has certain rights in this invention.

FIELD OF THE INVENTION

This invention pertains to the fields of immunodiagnostics andimmunotherapeutics. The invention provides methods of identifyinginternalizing antibodies and internalizing receptor ligands, as well asthe internalizing receptors bound.

BACKGROUND OF THE INVENTION

Growth factor receptors, and other signal transduction receptors, arefrequently overexpressed in human carcinomas and other diseases and thushave been utilized for the development of targeted therapeutics. TheHER2/neu gene, for example, is amplified in several types of humanadenocarcinomas, especially in tumors of the breast and the ovary(Slamon et al. (1989) Science 244: 707-712) leading to theoverexpression of the corresponding growth factor receptor ErbB2.Targeting of ErbB2 overexpressing cells has been accomplished primarilyusing anti-ErbB2 antibodies in different formats, including conjugationto liposomes containing chemotherapeutics (Kirpotin et al. (1997).Biochem. 36: 66-75), fusion to DNA carrier proteins delivering a toxicgene (Forminaya and Wels (1996) J. Biol. Chem. 271: 10560-10568), anddirect fusion to a toxin (Altenschmidt et al. (1997) Int. J. Cancer 73:117-124).

For many of these targeted approaches, it is necessary to deliver theeffector molecule across the cell membrane and into the cytosol. In somecases, this can be facilitated by taking advantage of receptor mediatedendocytosis (Ullrich and Schlessinger (1990) Cell 61: 203-212).Receptor-mediated endocytosis is often caused when ligand binding causesreceptor activation via homo- or heterodimerization, either directly forbivalent ligand or by causing a conformational change in the receptorfor monovalent ligand. Antibodies can mimic this process, stimulateendocytosis, become internalized and deliver their payload into thecytosol. In addition, the efficiency with which antibodies mediateinternalization differs significantly depending on the type of theantibody (e.g. whole antibody, fragment, single chain, monomeric,dimeric, etc.) and on the epitope recognized (Yarden (1990) Proc. Natl.Acad. Sci. USA 87: 2569-2573; Hurwitz et al. (1995) Proc. Natl. Acad.Sci. USA 92: 3353-3357.). Thus for some applications, such as liposomaltargeting, only antibodies that bind specific epitopes are rapidlyinternalized and yield a functional targeting vehicle.

Internalizing antibodies have also been shown to cause cell growthinhibition or enhanced cell growth, depending on the epitope recognized.Thus selection for internalization should lead to the isolation ofgrowth inhibitory or stimulatory (agonist) antibodies. Such inhibitoryantibodies might be used as cancer treatments or for the treatment ofother conditions characterized by cell hyperproliferation, and for thetreatment of inflammation (anti-inflammatories). Agonist antibodiescould be used for stimulating growth of relevant cells (for example stemcells). Targeting of cells besides cancer cells for gene delivery willalso have many application

Currently, antibodies that mediate internalization are identified byscreening hybridomas. Screening of hybridoma-produced antibodies,however, is laborious, time-consuming, and expensive.

SUMMARY OF THE INVENTION

This invention is based, in part, on the discovery that it is possibleto directly select internalizing antibodies from large non-immune phagelibraries by recovering infectious phage particles from within cellsafter receptor mediated endocytosis.

Thus, in one embodiment, this invention provides methods of selectingpolypeptide or antibody binding moieties that are internalized intotarget cells. The methods preferably involve i) contacting one or moreof target cells with one or more members of a phage display library; iv)culturing the target cells under conditions where members of the displaylibrary can be internalized if bound to an internalizing marker; and v)identifying internalized members of the phage display library if membersof the phage display library are internalized into one or more of thetarget cells. The methods also optionally, and preferably additionallyinvolve contacting members of the phage display library with a cells ofa subtractive cell line; and then washing the target cells to remove thecells of a subtractive cell line and to remove members of the phagedisplay library that are non-specifically bound or weakly bound to thetarget cells. In a preferred embodiment, the phage display library is anantibody phage display library, more preferably an antibody phagedisplay library displaying single chain antibodies (e.g. scFv, scFab,etc.).

In a preferred embodiment, the “identifying” step comprises recoveringinternalized phage and repeating steps the process again to furtherselect for internalizing binding moieties. In one embodiment, the“recovering” step involves lysing the target cells to releaseinternalized phage; and infecting a bacterial host with the internalizedphage to produce phage for a subsequent round of selection. Therecovering step can involve recovering infective phage, and/orrecovering a nucleic acid encoding a phage-displayed antibody and/orselection of phage expressing a selectable marker (e.g. an antibioticresistance gene or cDNA). The identifying step can involve detectingexpression of a reporter gene, detecting the presence absence orquantity of a particular nucleic acid, or selection of phage via aselectable marker. In preferred methods the cells of a subtractive cellline are present in at least 2-fold excess over the target cells. Inpreferred methods, the target cells form an adherent layer. In preferredmethods the target cell line is grown adherent to a tissue culture plateand co-incubated with the subtracting cell line in suspension in asingle cell culture flask. In particular preferred methods, thecontacting with a subtractive cell line is performed at a temperature(e.g. at about 4° C.) lower than the internalization culture conditions(e.g. at about 37°)

In particularly preferred embodiments, the phage express a selectablemaker and/or a reporter gene. Preferred selectable markers include, butare not limited to genes (or cDNAs) encoding fluorescent protein(s), anantibiotic resistance gene or cDNA, and a chromagenic gene or cDNA(e.g., horse radish peroxidase, β-lactamase, luciferase, andβ-galactosidase. In certain embodiments the target cells can includesolid tumor cells, members of a cDNA expression library, cells thatoverexpress a cytokine receptor, cells that overexpress a growth factorreceptor, metastatic cells, cells of a transformed cell line, cellstransformed with a gene or cDNA encoding a specific surface targetreceptor, and neoplastic cells derived from outside a solid tumor. Inone particularly preferred embodiment, the said cells of a subtractivecell line are selected from the same tissue type as the target cells.Suitable s subtractive cell line cells include, but are not limited tofibroblasts, monocytes, stem cells, and lymphocytes.

The methods of this invention can also be used to identify internalizingreceptors and/or internalizing receptor epitopes (regions of thereceptor that when bound induce internalization of the binding moiety).The methods generally involve any of the methods for identifyinginternalizing antibodies or polypeptides as described herein with theadditional steps whereby the internalizing antibodies or polypeptidesidentified are used to probe the original target cells, or differentcells. When the internalizing antibodies or polypeptides so bind, theypermit isolation of the cell bearing the internalizing receptor andisolation of the receptor and/or receptor epitope itself. Thus in oneembodiment the methods involve i) contacting one or more of the targetcells with one or more members of a phage display library; ii)optionally, but preferably, contacting members of the phage displaylibrary with a cells of a subtractive cell line; iii) optionally, butpreferably washing the target cells to remove said cells of asubtractive cell line and to remove members of the phage display librarythat are non-specifically bound or weakly bound to said target cells;iv) culturing the cells under conditions where members of said phagedisplay library can be internalized if bound to an internalizing marker;v) identifying internalized members of the phage display library ifmembers of the phage display library are internalized into one or moreof said target cells; vi) contacting the same or different target cellswith the identified internalized members of step (v) or memberspropagated therefrom, whereby the members bind to the surface of saidsame or different target cells. The method can further involve isolatinga component of the same or different target cells to which the membersbind. In some methods the “identifying” step involves recoveringinternalized phage and repeating steps (i) through (v) to further selectfor internalizing binding moieties.

The contacting, washing, culturing, and identifying steps are preferablyperformed as described herein and the target and subtractive cellsinclude the cells described herein.

In still another embodiment, this invention provides a multivalentantibody phage display library. The library preferably comprises aplurality of phage wherein the phage display, on average, at least twocopies of a single-chain antibody and the library comprises a pluralityof species of single-chain antibody. In preferred embodiments, the phagedisplay, on average, at least 3, at least 4, or at least 5 copies of asingle chain antibody per phage particle. Particularly preferredlibraries comprise, on average, at least 10⁵, preferably at least 10⁶,more preferably at least 10⁷, and most preferably at least 10⁸ differentspecies of single chain antibody. In a most preferred embodiment, theantibodies are encoded by a nucleic acids that are phage (not phagemid)vectors.

In certain embodiments, the library will be selected for members thatspecifically bind to an internalizing cell surface receptor (e.g. erbB2,EGF receptor, PDGF receptor, VEGF receptor, transferrin receptor, etc.).The single-chain antibodies are preferably single chain Fv (scFv) orsingle-chain Fab (scFab) antibodies. Filamentous phage are preferablyused in the libraries of this invention and the antibodies arepreferably expressed as a fusion with a PIII minor coat protein. Thephage can also express a selectable marker (e.g. an antibioticresistance gene or cDNA) and/or a reporter gene or cDNA (e.g., greenfluorescent protein (GFP), Fflux, β-gal, β-lactamase, etc.).

In still yet another embodiment, this invention provides a nucleic acidlibrary encoding one of the phage display antibody libraries describeherein. The nucleic acid library comprises at least 10⁵, more preferablyat least 10⁶, and most preferably at least 10⁷ different phage orphagemid vectors.

This invention also provides kits for practice of the methods describedherein. The kits preferably comprise one or more containers containing aphage display library (or a portion thereof) described herein. The kitcan include nucleic acids encoding the library and/or phage particlesexpressing single chain antibodies (preferably a multivalent library)and/or cells containing the intact phage or nucleic acids from thephage.

DEFINITIONS

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (scFv or scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked V_(H)-V_(L) heterodimer whichmay be expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker.Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Whilethe V_(H) and V_(L) are connected to each as a single polypeptide chain,the V_(H) and V_(L) domains associate non-covalently. The firstfunctional antibody molecules to be expressed on the surface offilamentous phage were single-chain Fv's (scFv), however, alternativeexpression strategies have also been successful. For example Fabmolecules can be displayed on phage if one of the chains (heavy orlight) is fused to g3 capsid protein and the complementary chainexported to the periplasm as a soluble molecule. The two chains can beencoded on the same or on different replicons; the important point isthat the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to g3p (see, e.g., U.S. Pat.No. 5,733,743). The scFv antibodies and a number of other structuresconverting the naturally aggregated, but chemically separated light andheavy polypeptide chains from an antibody V region into a molecule thatfolds into a three dimensional structure substantially similar to thestructure of an antigen-binding site are known to those of skill in theart (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778).Particularly preferred antibodies include all those that have beendisplayed on phage I think preferred antibodies should include all thathave been displayed on phage (e.g., scFv, Fv, Fab and disulfide linkedFv (Reiter et al. (1995) Protein Eng. 8: 1323-1331).

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (k_(on)) andthe “off rate constant” (k_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.The ratio of k_(off)/k_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the dissociation constant K_(d)(see, generally, Davies et al. (1990) Ann. Rev. Biochem., 59: 439-473.

The phrase “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. Specific binding to a proteinunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, F5 or C1 antibodiescan be raised to the c-erbB-2 protein that bind c-erbB-2 and not toother proteins present in a tissue sample. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are preferably in the natural “L” isomeric form. However,residues in the “D” isomeric form can be substituted for any L-aminoacid residue, as long as the desired functional property is retained bythe polypeptide. In addition, the amino acids, in addition to the 20“standard” amino acids, include modified and unusual amino acids, whichinclude, but are not limited to those listed in 37 CFR □1.822(b)(4).Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates either a peptide bond to afurther sequence of one or more amino acid residues or a covalent bondto a carboxyl or hydroxyl end group.

The term “binding polypeptide” refers to a polypeptide that specificallybinds to a target molecule (e.g. a cell receptor) in a manner analogousto the binding of an antibody to an antigen. Binding polypeptides aredistinguished from antibodies in that binding polypeptides are notultimately derived from immunoglobulin genes or fragments ofimmunoglobulin genes.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated.

Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka etal. (1985) J. Biol. Chem. 260: 2605-2608; and Cassol et al. (1992);Rossolini et al., (1994) Mol. Cell. Probes 8: 91-98). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

The terms “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state. However, the term “isolated”is not intended refer to the components present in an electrophoreticgel or other separation medium. An isolated component is free from suchseparation media and in a form ready for use in another application oralready in use in the new application/milieu.

A chimeric molecule is a molecule in which two or more molecules thatexist separately in their native state are joined together to form asingle molecule having the desired functionality of all of itsconstituent molecules. While the chimeric molecule may be prepared bycovalently linking two molecules each synthesized separately, one ofskill in the art will appreciate that where the chimeric molecule is afusion protein, the chimera may be prepared de novo as a single “joined”molecule.

A fusion protein is a chimeric molecule in which the constituentmolecules are all polypeptides and are attached (fused) to each otherthrough terminal peptide bonds so that the chimeric molecule is acontinuous single-chain polypeptide. The various constituents can bedirectly attached to each other or can be coupled through one or morepeptide linkers.

An effector moiety or molecule is a molecule or moiety that typicallyhas a characteristic activity that is desired to be delivered to atarget cell (e.g. a tumor overexpressing c-erbB-2). Effector moleculesinclude cytotoxins, labels, radionuclides, ligands, antibodies, drugs,liposomes, and viral coat proteins that render the virus capable ofinfecting a c-erbB-2 expressing cell.

A “target” cell refers to a cell or cell-type that is to be specificallybound by a member of a phage display library or a chimeric molecule ofthis invention. Preferred target cells are cells for which aninternalizing antibody or binding polypeptide is sought. The target cellis typically characterized by the expression or overexpression of atarget molecule that is characteristic of the cell type. Thus, forexample, a target cell can be a cell, such as a tumor cell, thatoverexpresses a marker such as c-erbB-2.

A “targeting moiety” refers to a moiety (e.g. a molecule) thatspecifically binds to the target molecule. Where the target molecule isa molecule on the surface of a cell and the targeting moiety is acomponent of a chimeric molecule, the targeting moiety specificallybinds the chimeric molecule to the cell bearing the target. Where thetargeting moiety is a polypeptide it can be referred to as a “targetingpolypeptide”.

The terms “internalizing” or “internalized” when used in reference to acell refer to the transport of a moiety (e.g. phage) from outside toinside a cell. The internalized moiety can be located in anintracellular compartment, e.g. a vacuole, a lysosome, the endoplasmicreticulum, the golgi apparatus, or in the cytosol of the cell itself.

An internalizing receptor or marker is a molecule present on theexternal cell surface that when specifically bound by an antibody orbinding protein results in the internalization of that antibody orbinding protein into the cell. Internalizing receptors or markersinclude receptors (e.g., hormone, cytokine or growth factor receptors)ligands and other cell surface markers binding to which results ininternalization.

The term “heterologous nucleic acid’ refers to a nucleic acid that isnot native to the cell in which it is found or whose ultimate origin isnot the cell or cell line in which the “heterologous nucleic acid” iscurrently found.

The idiotype represents the highly variable antigen-binding site of anantibody and is itself immunogenic. During the generation of anantibody-mediated immune response, an individual will develop antibodiesto the antigen as well as anti-idiotype antibodies, whose immunogenicbinding site (idiotype) mimics the antigen. Anti-idiotypic antibodiescan also be generated by immunization with an antibody, or fragmentthereof.

A “phage display library” refers to a collection of phage (e.g.,filamentous phage) wherein the phage express an external (typicallyheterologous) protein. The external protein is free to interact with(bind to) other moieties with which the phage are contacted. Each phagedisplaying an external protein is a “member” of the phage displaylibrary.

An “antibody library” refers to phage display library that displaysantibodies (binding proteins encoded by one or more antibody genes orcDNAs). The antibody library includes the population of phage or acollection of vectors encoding such a population of phage, or cell(s)harboring such a collection of phage or vectors. The library can bemonovalent, displaying on average one single-chain antibody per phageparticle or multi-valent displaying, on average, two or more singlechain antibodies per viral particle. Preferred antibody librariescomprise on average more than 10⁶, preferably more than 10⁷, morepreferably more than 10⁸, and most preferably more than 10⁹ differentmembers (i.e. encoding that many different antibodies).

The term “filamentous phage” refers to a viral particle capable ofdisplaying a heterogenous polypeptide on its surface. Although oneskilled in the art will appreciate that a variety of bacteriophage maybe employed in the present invention, in preferred embodiments thevector is, or is derived from, a filamentous bacteriophage, such as, forexample, f1, fd, Pf1, M13, etc. The filamentous phage may contain aselectable marker such as tetracycline (e.g., “fd-tet”). Variousfilamentous phage display systems are well known to those of skill inthe art (see, e.g., Zacher et al. (1980) Gene 9: 127-140, Smith etal.(1985) Science 228: 1315-1317 (1985); and Parmley and Smith (1988)Gene 73: 305-318).

A “viral packaging signal” is a nucleic acid sequence necessary andsufficient to direct incorporation of a nucleic acid into a viralcapsid.

An assembly cell is a cell in which a nucleic acid can be packaged intoa viral coat protein (capsid). Assembly cells may be infected with oneor more different virus particles (e.g. a normal or debilitated phageand a helper phage) that individually or in combination direct packagingof a nucleic acid into a viral capsid.

The term “detectable label” refers to any material having a detectablephysical or chemical property. Such detectable labels have beenwell-developed in the field of immunoassays and, in general, any labeluseful in such methods can be applied to the present invention. Thus, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texasred, rhodamine, and the like), radiolabels (e.g., 3H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., LacZ, CAT, horse radish peroxidase, alkalinephosphatase and others, commonly used as detectable enzymes, either asmarker gene products or in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads. Those detectable labels that can beexpressed by nucleic acids are referred to as “reporter genes” or“reporter gene products”.

It will be recognized that fluorescent labels are not to be limited tosingle species organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe-CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

The following abbreviations are used herein: AMP, ampicillin; c-erbB-2ECD, extracellular domain of c-erbB-2; CDR, complementarity determiningregion; ELISA, enzyme linked immunosorbent assay; FACS, fluorescenceactivated cell sorter; FR, framework region; Glu, glucose; HBS, hepesbuffered saline, 10 mM hepes, 150 mM NaCl, pH 7.4; IMAC, immobilizedmetal affinity chromatography; k_(on), association rate constant;k_(off), dissociation rate constant; MPBS, skimmed milk powder in PBS;MTPBS, skimmed milk powder in TPBS; PBS, phosphate buffered saline, 25mM NaH₂PO₄, 125 mM NaCl, pH 7.0; PCR, polymerase chain reaction; RU,resonance units; scFv or scFv, single-chain Fv fragment; TPBS, 0.05% v/vTween 20 in PBS; SPR, surface plasmon resonance; V_(k), immunoglobulinkappa light chain variable region; V_(λ), immunoglobulin lambda lightchain variable region; V_(L), immunoglobulin light chain variableregion; V_(H), immunoglobulin heavy chain variable region; wt, wildtype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the method for construction of a large human scFvphage antibody library. The strategy for library construction involvedoptimizing the individual steps of library construction to increase boththe efficiency of scFv gene assembly and to increase the efficiency ofcloning assembled scFv genes. (A). First, mRNA from lymphocytes was usedto generate V_(H) and V_(L) gene repertoires by RTPCR which were clonedinto different vectors to create V_(H) and V_(L) gene libraries of8.0×10⁸ and 7.2×10⁶ members respectively. The cloned V-gene librariesprovided a stable and limitless source of V_(H) and V_(L) genes for scFvassembly. DNA encoding the peptide (G₄S)₃ was incorporated into the 5′end of the V_(L) library. This permitted generation of scFv genes by PCRsplicing 2 DNA fragments. Previously, scFv gene repertoires wereassembled from 3 separate DNA fragments consisting of V_(H), V_(L), andlinker DNA. (B) V_(H) and V_(L) gene repertoires were amplified from theseparate libraries and assembled into an scFv gene repertoire usingoverlap extension PCR. The primers used to reamplify the V_(H) and V_(L)gene repertoires annealed 200 bp upstream of the 5′ end of the V_(H)genes and 200 bp down stream of the V_(L) genes. These long overhangsensured efficient restriction enzyme digestion.(C.) The scFv generepertoire was digested with NcoI and NotI and cloned into the plasmidpHEN1 as fusions with the M13 gene III coat protein gene ( ) forphage-display.

FIGS. 2A 2B, and 2C show schematics illustrating antibody phage display:Cartoon of phage displaying (2A) a single scFv (2B) a single diabody or(2C) multiple scFv. scFv=single chain Fv antibody fragment; V_(H)=Igheavy chain variable domain; V_(L)=Ig light chain variable domain;pIII=phage minor coat protein pIII; Ag=antigen bound by scFv.

FIG. 3 shows the effect of trypsinization on the enrichment of antigenspecific phage. A mixture of fd phage (5.0×10¹¹ cfu) and C6.5 scFvphagemid (5.0×10⁸ fu) was incubated with SKBR3 cells for 2 hours at 37°C. Washes were performed either as described in Table 6 (−) or cellswere trypsinized prior to cell lysis (+). Phage present in the firststripping buffer wash (cell surface phage) and the cell lysate(intracellular phage) were titered in the presence of ampicillin (C6.5phagemid) or tetracycline (fd phage).

FIG. 4 shows the effect of incubation time and chloroquine on therecovery of antigen specific phage. SKBR3 cells were incubated in thepresence (▪, ) or absence (□, ◯) of chloroquine (50 μM) for 2 hoursprior to the addition of anti-botulinum phagemid (□, ▪) or C6.5 scFvphagemid (◯, ) (1.5×10⁹ cfu/ml). Cell samples were taken at 0 minutes,20 minutes, 1 hour or 3 hours after phage addition, washed as describedin FIG. 4 including the trypsinization step and intracellular phagestitered.

FIG. 5 shows the effect of phage concentration on the recovery ofintracellular phage. Various concentrations of C6.5 scFv phagemid,C6ML3-9 scFv phagemid, C6.5 diabody phagemid or C6.5 scFv phage (inputphage titer) were incubated with subconfluent SKBR3 cells grown in6-well plates for 2 hours at 37° C. Cells were treated as described inFIG. 4 including the trypsinization step and intracellular phage weretitered (output phage titer).

FIG. 6 illustrates strategies for producing anti-ErbB2 phagemids andphages packaging a eukaryotic reporter gene. Left column: Helper phageare used to infect TG1 containing pHEN-F5-GFP, a phagemid composed of anf1 origin of replication (f1 ori), the anti-ErbB2 F5 scFv gene fused togene III and an eukaryotic GFP reporter gene driven by the CMV promoter.Phage recovered from the culture supernatant display an average of 1scFv-pIII fusion protein and 99% of them package the GFP reporter gene.Right column: the anti-ErbB2 F5 scFv gene is cloned into the fd phagegenome for expression as a scFv-pIII fusion. fd-F5 phages are used toinfect TG1 containing a GFP reporter phagemid vector (pcDNA3-GFP).Phages purified from the culture supernatant display multiple scFv-pIIIfusion protein and approximately 50% package the GFP reporter gene.

FIG. 7 shows a comparison of anti-ErbB2 phagemid and phage binding oncells. 10⁵ ErbB2 expressing SKBR3 cells were incubated with increasingconcentrations of F5-phagemids (circles) or fd-F5-phages (squares) at 4°C. for 1 hour. Cell surface bound phages were detected with biotinylatedanti-M13 and streptavidin-PE. Binding was detected by FACS and theresults expressed as mean fluorescent intensity (MFI).

FIGS. 8A and 8B illustrate phagemid-mediated gene transfer in breastcancer cell lines. (FIG. 8A) (1, 2, 3) 2.0×10⁵ MCF7 (low ErbB2expression) or (4, 5, 6) 2.0×10⁵ SKBR3 (high ErbB2 expression) cellsgrown in 6-well plates were incubated with either no (1,4) no phage, (2,5) 5.0×10¹² cfu/ml of helper phage packaging GFP or (3, 6) 5.0×10¹¹cfu/ml of F5-GFP-phagemids for 48 hrs. Cells were trypsinized and GFPdetected by FACS. (FIG. 8B) An equal number of MCF7 and SKBR3 cells(1.0×10⁵) were grown together and incubated with 5.0×10¹¹ cfu/ml ofF5-GFP-phagemids for 48 hrs. Cells were trypsinized and stained forErbB2 expression using 4D5 antibody and rhodamine conjugated sheepanti-mouse Ig to discriminate SKBR3 (Region R1) and MCF7 (Region R2)cells. The GFP content of each subpopulation was determined by FACS.

FIGS. 9A, 9B, 9C, and 9D show concentration dependence and time courseof phagemid mediated GFP expression in SKBR3 cells. FIGS. 9A and 9B showconcentration dependence of phagemid and phage mediated GFP expressionin SKBR3 cells. 5.0×10⁴ cells were grown in 24-well plates and incubatedwith increasing concentrations of F5-GFP-phagemid (squares),fd-F5-GFP-phage (diamonds) or GFP-helper phage (circles). After 60 hrs,the cells were trypsinized and analyzed by FACS for GFP expression.FIGS. 9C and 9D show the time dependence of phagemid mediated GFPexpression in SKBR3 cells. 5.0×10⁴ cells were incubated with 5.10¹¹cfu/mL of F5-GFP-phagemid and analyzed for GFP expression by FACS. Forincubation times greater than 48 hrs, the phage were added to 2.5×10⁴cells and the culture medium was replaced by fresh medium after 48 hrsof incubation. The results are expressed as (9A, 9C) % of GFP positivecells and (9B, 9D)) MFI of the GFP positive cells.

DETAILED DESCRIPTION I. Introduction

This invention provides new methods of screening for specific bindingpolypeptides and/or antibodies that are internalized by particulartarget cells. Unlike prior art assay methods that simply detect bindingto an external target on a cell (e.g. a receptor) the assays of thisinvention explicitly identify molecules that bind and are transportedinto the cell (i.e. into a vacuole and/or the endoplasmic reticulumand/or into the cytosol itself).

The utility of a number of specific antibodies, even those generallyknown to bind to internalizing receptors (e.g. c-erbB-2) has beenlimited by the frequent lack of internalization of the bound antibody orby unacceptably slow internalization rates. Such antibodies while usefulfor delivering moieties to the cell surface, have proven generallyunsatisfactory of the delivery of effector molecules that must obtainentry into the cell for activity.

In contrast, the binding polypeptides and/or antibodies identified bythe methods of this invention are rapidly internalized into the cell.They are thus extremely useful for delivering effector moieties into thetarget cell. Moreover, once an internalizing antibody or polypeptide isidentified it can be used to re-probe one or more cells or cell lines toidentify previously unknown internalizing cellular targets (e.g.,receptors).

In addition, selecting for internalization also selects for biologicfunction. Many receptors (for example growth factor receptors) useinternalization as a way of modulating and regulating the effect ofligands. For example, ligand binding can result in signal transductionand receptor internalization. The decrease in the number of receptorsthen causes down regulation of the effect of additional ligand. The sameoccurs with antibodies that bind growth factor receptors (Hurwitz et al.(1995) Proc. Natl. Acad. Sci. USA. 92: 3353-3357). For example,“[g]rowth factors act by binding to and activating the intrinsiccatalytic activity of their cell surface receptors, thereby initiating asignaling cascade leading to the cellular response. Growthfactor/receptor complexes are not static residents of the cell surfacemembrane but undergo endocytotic trafficking processes ofinternalization and sorting to recycling or degradation. Consequently,growth factors are depleted from the extracellular medium and theirreceptors undergo down-regulation. These trafficking processes, byvirtue of their influence on the kinetics of signaling growthfactor/receptor complexes, are important modulators of cell behavioralresponses” (Reddy et al. (1996) Nature Biotech. 14: 1696-1699)

In the ErbB2 system, one mechanism by which ErbB2 binding antibodiesinhibit growth is to cause receptor internalization and down regulation(Hurwitz et al. (1995) Proc. Natl. Acad. Sci. USA. 92: 3353-3357). Italso may be possible to turn an internalizing antibody that binds agrowth factor receptor and causes growth inhibition into a growthstimulatory antibody. For example, the mitogenic properties of EGF havebeen increased by lowering the affinity of EGF for the EGF receptor. Thelower affinity EGF causes receptor signaling, but reducedinternalization and down regulation than wild type EGF (presumably fromthe lower affinity) (Reddy et al. (1996) Nature Biotech. 14: 1696-1699).Thus lowering the affinity of a growth inhibiting internalizing antibodycould turn it into a growth factor. Thus identification of internalizingantibodies can provide lead compounds/drugs for both growth inhibitionand growth stimulation.

II. Methods of Identifying Internalizing Antibodies and/or Receptors

A) Identification of Internalizing Polypeptides/Antibodies.

In one embodiment, this invention provides methods for identifyinginternalizing antibodies or polypeptides. The methods involve contactinga “target” cell with one or more members of a phage display librarydisplaying an antibody or a binding polypeptide. The phage displaylibrary is preferably a multivalent phage display library and it isbelieved that this invention provides the first description of amultivalent antibody phage display library.

After a suitable incubation period, the cells are washed to removeexternally bound phage (library members) and then internalized phage arereleased from the cells, e.g., by cell lysis. It was a discovery of thisinvention that the internalized phage are still viable (infectious).Thus the internalized phage in the cell lysate can be recovered andexpanded by using the lysate containing internalized phage to infect abacterial host. Growth of infected bacteria leads to expansion of thephage which can be used for a subsequent round of selection. Each roundof selection enriches for phage which are more efficiently internalized,more specific for the target cell or have improved bindingcharacteristics.

The phage display library is preferably contacted with a subtractivecell line (i.e. a subtractive cell line is added to the target cells andculture media) to remove members of the phage display library that arenot specific to the “target” cell(s). The subtractive cell line ispreferably added under conditions in which members of the phage displaylibrary are not internalized (e.g., at a temperature of about 4° C. toabout 20° C., more preferably at a temperature of about 4° C.) so thatnon-specific binding members of the library are not internalized(sequestered) before they can be subtracted out by the subtractive cellline.

After subtracting out non-specific binding antibodies, the “target”cells are washed to remove the subtractive cell line and to removenon-specifically or weakly-bound phage.”

The target cells are then cultured under conditions where it is possiblefor internalization to occur (e.g. at a temperature of about 35° C. toabout 39° C., more preferably at a temperature of about 37° C.). Theduration of the internalization culture period will determine theinternalization speed of the antibodies (phage display members) forwhich selection takes place. With shorter internalization periods morerapid internalizing antibodies are selected while with longerinternalization periods slower internalizing antibodies are selected.The internalization period is preferably less than about 120 minutes,more preferably less than about 60 minutes, and most preferably lessthan about 30 minutes or even less than about 20 minutes.

It is noted that during the internalization period the target cells aregrown under conditions in which internalization can occur. For a numberof cell lines, this involves culturing the cells adherently on cultureplates.

After internalization has been allowed to occur the target cells arewashed to remove non-internalized (e.g. surface-bound phage).

The cells can then be moved to clean media. In a preferred embodiment,where the cells are adherent, they cells are trypsinized to free thecells from the extracellular matrix which may contain phage antibodiesthat bind the extracellular matrix. Freeing the cells into solutionpermits more through washing and moving of the cells to a new cultureflask will leave behind any phage that may have stuck to the tissueculture dish.

The cells can then be washed with a large volume of PBS and lysed torelease the internalized phage which can then be expanded e.g. used toinfect E. coli to produce phage for the next round of selection. It isnoted that there is no need to actually visualize the internalizedphage. Simple cell lysis and expansion of the formerly internalizedphage is sufficient for recovering internalizing phage display members.

B) Identification of Internalizing Receptors.

Once an antibody or polypeptide that is internalized into a cell hasbeen identified, it is possible to probe one or more cell types with theidentified antibody or polypeptide to identify the target recognized andbound by the antibody. Since the antibody is an internalizing antibodyit is likely that such targets are themselves internalizing targets(e.g. members or portions of internalizing receptors).

In one embodiment, the antibody can be labeled as described below. Thecells can then be contacted with the antibody (i.e. in vivo or in vitro)and the cells or cellular regions to which the antibody binds can thenbe isolated.

Alternatively, the antibodies can be used e.g. in an affinity matrix(e.g. affinity column) to isolate the targets (e.g. receptor or receptorsubunits) to which they bind. Briefly, in one embodiment, affinitychromatography involves immobilizing (e.g. on a solid support) one ormore species of the internalizing antibodies identified according to themethods of this invention. Cells, cellular lysate, or cellularhomogenate are then contacted with the immobilized antibody which thenbinds to its cognate ligand. The remaining material is then washed awayand the bound/isolated cognate ligand can then be released from theantibody for further use. Methods of performing affinity chromatographyare well known to those of skill in the art (see, e.g., U.S. Pat. Nos.5,710,254, 5,491,096, 5,278,061, 5,110,907, 4,985,144, 4,385,991,3,983,001, etc.).

In another embodiment, the antibodies are used to immunoprecipitate thetarget from cell lysate. The precipitate is then run on an SDS-PAGE gelwhich is Western blotted onto nitrocellulose. The blot is probed withthe precipitating antibody to identify the location of the target. Theportion of the blot containing the target can then be sent forN-terminal protein sequencing. The N-terminal sequence can then be usedto identify the target from standard databases, or DNA probes can besynthesized to probe genomic or cDNA libraries. This approach has beenused to identify the antigen bound by a phage antibody. Selections of aphage antibody library were done on intact Chlamydia trachomatis (abacterial like organism that causes Chlamydial diseases). Selectedantibodies were then used as described above to identify the antigenbound.

C) Functional Genomics

In another embodiment, the assays of this invention can be used toscreen libraries to identify previously unknown binding agents. Thereare two preferred approaches to this proteomic or functional genomicanalysis. In the first, a phage displayed cDNA library is created. mRNA(probably subtracted) is made from the cell line or tissue of interest.First strand DNA is synthesized and treated with DNAse or fragmented insome other way. This removes the 5′ and 3′ UTR and the 3′ stop codon. Aphage library is then produced and selected for internalization oncells.

Ligands (or domains of ligands) that bind to cell surface receptors andinternalized are identified. This approach might be used, for example,to identify orphan growth factors which bind to internalizing growthfactor receptors. If a receptor is known, but the ligand is not, thereceptor gene could be transfected into a cell line and the transfectedcell line used for selection. The selection could also be combined withdelivery of a reporter gene. In this case, the phage vector that is usedto create the phage library would contain the reporter gene. Afterselection, one could isolate for example, green cells expressing thereporter gene GFP by FACS (rather than lysing all cells to recoverinternalized phage). This is expected to improve the specificity ofselection.

For the second approach, second paragraph: the phage library is selectedfor internalization on a target cell line as described above. Theselected polyclonal or monoclonal phage are then used to flow sort cells(e.g. COS cells) transfected with a cDNA library. cDNA library plasmidsare recovered from sorted cells and amplified in bacteria using standardtechniques. The amplified plasmid cDNA library is used to transfectcells (e.g., COS cells) which are again sorted using phage. Afterseveral rounds of selection, sorted cells should contain only plasmidsencoding cell surface receptors bound by internalizing phage. These canbe identified by DNA sequencing, and by testing each plasmid cDNA forbinding of phage after the plasmid DNA is used to transfect COS cells.

III. Assay Components

A) Phage Display Library.

1) Mono-Valent Antibody Libraries and Polypeptide Libraries.

The ability to express polypeptide and antibody fragments on the surfaceof viruses which infect bacteria (bacteriophage or phage) makes itpossible to isolate a single binding polypeptide or antibody fragmentfrom a library of greater than 10¹⁰ nonbinding clones. To expresspolypeptide or antibody fragments on the surface of phage (phagedisplay), a polypeptide or an antibody fragment gene is inserted intothe gene encoding a phage surface protein (pIII) and the antibodyfragment-pIII fusion protein is displayed on the phage surface(McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991)Nucleic Acids Res. 19: 4133-4137). Since the antibody fragments on thesurface of the phage are functional, phage bearing antigen bindingpolypeptides or antibody fragments can be separated from non-bindingphage by antigen affinity chromatography (McCafferty et al. (1990)Nature, 348: 552-554). Depending on the affinity of the antibodyfragment, enrichment factors of 20 fold-1,000,000 fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000 fold in one roundcan become 1,000,000 fold in two rounds of selection (McCafferty et al.(1990) Nature, 348: 552-554). Thus even when enrichments are low (Markset al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen after four rounds of selection. Thusonly a relatively small number of clones (several hundred) need to beanalyzed for binding to antigen.

In a preferred embodiment, analysis for binding is simplified byincluding an amber codon between the antibody fragment gene and geneIII. The amber codon makes it possible to easily switch betweendisplayed and soluble (native) antibody fragment simply by changing thehost bacterial strain (Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137).

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In the first Example, naturalV_(H) and V_(L) repertoires present in human peripheral bloodlymphocytes were isolated from unimmunized donors by PCR. The V-generepertoires were spliced together at random using PCR to create a scFvgene repertoire which was cloned into a phage vector to create a libraryof 30 million phage antibodies (Id.). From this single “naive” phageantibody library, binding antibody fragments have been isolated againstmore than 17 different antigens, including haptens, polysaccharides andproteins (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al.(1993). Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies havebeen produced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). It is also possible to isolate antibodies againstcell surface antigens by selecting directly on intact cells. Forexample, antibody fragments against four different erythrocyte cellsurface antigens were produced by selecting directly on erythrocytes(Marks et al. (1993). Bio/Technology. 10: 779-783). Antibodies wereproduced against blood group antigens with surface densities as low as5,000 sites/cell. The antibody fragments were highly specific to theantigen used for selection, and were functional in agglutination andimmunofluorescence assays. Antibodies against the lower density antigenswere produced by first selecting the phage antibody library on a highlyrelated cell type which lacked the antigen of interest. This negativeselection removed binders against the higher density antigens andsubsequent selection of the depleted phage antibody library on cellsexpressing the antigen of interest resulted in isolation of antibodiesagainst that antigen. With a library of this size and diversity, atleast one to several binders can be isolated against a protein antigen70% of the time. The antibody fragments are highly specific for theantigen used for selection and have affinities in the 1 M to 100 nMrange (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Griffiths et al.(1993) EMBO J. 12: 725-734). Larger phage antibody libraries result inthe isolation of more antibodies of higher binding affinity to a greaterproportion of antigens.

The creation of a suitable large phage display antibody library isdescribed in detail in Example 1.

2) Polyvalent Antibody Phage Display Libraries

The probability of selecting internalizing antibodies from aphage-display antibody library is increased by increasing the valency ofthe displayed antibody. This approach takes advantage of normalcell-surface receptor biology. Often cell-surface receptors (e.g. growthfactor receptors) activate upon binding their cognate ligand through aprocess of homo- or heterodimerization (or trimerization, ortetramerization, etc.). The association of the receptor subunits in thisprocess can be mediated directly (e.g. when bound by a bivalent ligand)or indirectly by causing a conformational change in the receptor.

It was a discovery of this invention that polyvalent antibodies in adisplay library (e.g. a phage display library) can mimic this process,stimulate endocytosis, become internalized and deliver their payloadinto the cytosol. Thus, to increase the likelihood of identifyinginternalizing antibodies or recognizing internalizing epitopes,preferred embodiments of this invention utilize a polyvalent phagedisplay antibody library. It is believed that no multivalentphage-display antibody libraries have been created prior to thisinvention. Unlike the multivalently displayed peptide phage libraries,phage antibody libraries typically display monomeric single chain Fv(scFv) or Fab antibody fragments fused to pIII as single copies on thephage surface using a phagemid system (Marks et al. (1991) J. Mol. Biol.222: 581-597; Sheets et al. (1998) Proc. Natl. Acad. Sci. USA 95:6157-6162.).

As used herein, a polyvalent phage display antibody library, refers to alibrary in which each member (e.g. phage particle) displays, on average)two or more binding domains, wherein each binding domain includes avariable heavy and a variable light region. More generally, amultivalent phage display library displays, on average, two or more pIllfusions per page particle. Polyvalent phage display can be achieved byexpressing diabodies (i.e., a protein formed by fusion or conjugation oftwo single chain antibodies (e.g. scFv)) or by display of, on average,two or more antibodies on each phage particle. In contrast, amono-valent library displays, on average, one single-chain antibody perviral particle.

a) Diabody Expression.

Diabodies are scFv dimers where each chain consists of heavy (V_(H)) andlight (V_(L)) chain variable domains connected using a linker (e.g. apeptide linker) that is too short to permit pairing between domains onthe same chain. Consequently, pairing occurs between complementarydomains of two different chains, creating a stable noncovalent dimerwith two binding sites (Holliger et al. (1993) Proc. Natl. Acad. Sci.90: 6444-6448). The C6.5 diabody was constructed by shortening thepeptide linker between the Ig V_(H) and V_(L) domains from 15 to 5 aminoacids and binds ErbB2 on SKBR3 cells bivalently with a K_(d)approximately 40 fold lower than C6.5 (4.0×10⁻¹⁰ M) (Adams et al. (1998)Brit. J. Cancer. 77: 1405-1412, 1998).

In Example 5, described herein, C6.5 diabody genes were subcloned forexpression as pIII fusions in the phagemid pHEN-1 (Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133-4137). This yielded phagemidpredominantly expressing a single scFv or diabody-pIII fusion afterrescue with helper phage (Marks et al. (1992) J. Biol. Chem. 267:16007-16010). Diabody phagemid display a bivalent antibody fragmentresulting from intermolecular pairing of one scFv-pIII fusion moleculeand one native scFv molecule. Using the teachings provided herein one ofskill in the art can routinely produce other diabodies.

Phage displaying bivalent diabodies or multiple copies of scFv were moreefficiently endocytosed than phage displaying monomeric scFv andrecovery of infectious phage was increased by preincubation of cellswith chloroquine.

The results indicate that it is possible to select for endocytosableantibodies, even at the low concentrations that would exist for a singlephage antibody member in a library of 10⁹ members.

b) Polyvalent Display of Single-Chain Antibodies.

As an alternative to the use of diabodies, antibody phage displaylibraries are created in which each viral particle, on average,expresses at least 2, preferably at least 3, more preferably at least 4,and most preferably at least 5 copies of a single chain antibody.

In principle, each copy of pIII on the page (and there is controversy asto whether there are 3 or 5 copies of pIII per phage) should express anantibody. However, proteolysis occurs and the number actually displayedis typically less. Thus, preferred multivalent antibody libraries areconstructed in a phage vector and not a phage mid vector. This meansthat helper phage need not be added to make phage. Helper phage bringinto the E. coli wild-type pIII that competes with the scFv-pIII fusion.Thus, in phagemid vector, this competition leas, on average, to only 1(ore less) antibody per phage.

To produce multivalent antibody libraries, the single chain antibodies,typically expressed in phagemid, are subcloned from the phagemid vectorinto a phage vector. No helper phage is required and there is nocompetition between the wild-type pIII and the fusion scFv pIII fusion.thus, on average, the phage display two or more pill fusions. Thus, byway of illustration, Example 5 describes the subcloning of the C6.5 scFvgene into the phage vector fd-Sfi/Not. This results in phage with 3 to 5copies each of scFv-pIII fusion protein.

B) Target Cells.

The target cells of this invention include any cell for which it isdesired to identify an internalizing polypeptide or antibody or forwhich it is desired to identify an internalizing marker (e.g. receptor).The cells can include cells of multicellular eukaryotes, uni-cellulareukaryotes, including plants and fungi, and even prokaryotic cells.Preferred target cells are eukaryotic, more preferably vertebrate cells,and most preferably mammalian cells (e.g. cells from murines, bovines,primates including humans, largomorphs, canines, felines, and so forth).The cells can be normal healthy cells or cells characterized by aparticular pathology (e.g. tumor cells).

Target cells can include any cell type where it would be useful to: 1)have an antibody specifically recognize the cell type or related celltypes (for example for cell sorting, cell staining or other diagnosticprocedures); 2) have a ligand which is specifically internalized intothe cell type or related cell types (for example to deliver a toxic ortherapeutic gene or protein). Additional target cells include, but arenot limited to differentiated cells (i.e. differentiated to become atissue, e.g. prostate, breast). Thus an antibody that recognized andkilled prostate cells would be good for prostate cancer even if itkilled normal prostate cells (the prostate is not an essential organ).Target cells may include tissue specific cells, and cells at a givendevelopmental stage. Target cells may also include precursor cells, e.g.bone marrow stem cells, would be useful for isolating, perhapsstimulating for differentiation.

Target cells can also include cell lines transfected with a gene for aknown receptor (for example ErbB2) to which it would be useful to haveinternalizing antibodies.

Many ErbB2 antibodies are not internalizing. Rather than immunizing withrecombinant protein or selecting a phage library on recombinant protein,selection on ErbB2 transfected cells for internalization should yieldprecisely antibodies with the desired characteristics (internalization).Finally, a cDNA library could be transfected into a cell line (forexample COS) from a desired target cell line or tissue and phageantibodies selected for internalization. After several rounds ofselection, the phage could be used to stain and sort (for example byFACS) transfected cells. DNA can be recovered from the cells, yieldingthe sequences of internalizing receptors as well as phage antibodiesthat bind them.

C) Cells of a Subtractive Cell Line.

In a preferred embodiment of the assays of this invention, the phagedisplay library is contacted with cells from a “subtractive” cell line.This step is intended to deplete or eliminate members of the phagedisplay library that either bind the cells non-specifically or that bindto targets other than the target against which it is desired to obtain abinding polypeptide or antibody. The contacting with the cells from a“subtractive” cell line can occur before, during, or after the targetcells are contacted with members of the phage display library. However,in a preferred embodiment, the contacting with cells of a subtractivecell line is simultaneous with contacting of the target cells. Thus, forexample, in a preferred embodiment the target cell line (grown adherentto a tissue culture plate) is co-incubated with the subtracting cellline (in suspension) in a single cell culture flask.

Virtually any cell can act as a subtractive cell. However, in apreferred embodiment, subtractive cells display all the markers on thetarget cell except the marker (e.g. receptor) that is to act as a targetfor selection of the desired binding antibodies or binding polypeptides.Particularly preferred cells are thus closely related to the targetcell(s), in terms of having common internalizing cell surface receptors(such as transferring for example fibroblasts. If one was selecting on atumor cell line (for example a breast tumor cell line), than one couldnegatively select on a normal breast cell line. This may, however,deplete for antibodies that bind to overexpressed antigens, so again aparallel path would be to negatively select on fibroblasts. If one wasusing transfected cells, than non-transfected cell could be used as thesubtractive cell line. Where the tumor is epithelial in origin, thepreferred subtractive cell will also be epithelial and even morepreferably from the same tissue or organ.

Particularly preferred subtractive cells include, but are not limitedto, non-differentiated cell lines, non-transfected cells, mixtures ofnon-differentiated and non-transfected cells. When selecting forinternalization on tumor cells, preferred subtractive cell lines arepreferably the non-tumor cells of the same tissue (for example, breasttumor cells versus normal breast epithelial cells). Also, for cDNAexpression libraries, the subtractive cell line will be thenon-transformed cell line used for library construction (e.g. COS, CHO,etc.).

In one particularly preferred embodiment, the “target” cell is a celltransformed with a gene or cDNA for a specific target receptor. In thisinstance, the subtractive cell line is preferably the non-transformedcell line. Thus for example where CHO cells are transformed with avector containing the gene for the EGF receptor, the EGF-expressingcells are used as the target cell line, and the subtractive cell line isthe untransformed CHO cells. Using this approach internalizing anti-EGFreceptor antibodies were obtained.

The subtractive cells are more effective when provided in excess overthe target cells. The excess is preferably at least about a 2-fold toabout a 1000-fold excess, more preferably about a 3-fold to about a100-fold excess, and most preferably about a 5-fold to about a 50-foldexcess. In one embodiment, a 5-fold excess is sufficient.

D) Washing Steps.

As indicated above a variety of washing steps are used in the methods ofthis invention. In particular, a “weak” washing step can be used toremove the subtractive cells and weakly or non-specifically bindingmembers of the phage display library. A second strong washing step ispreferably used after internalization of members of the phage displaylibrary. The “strong” washing step is intended to remove tightly- andweakly-bound surface phage.

Buffers and methods for performing weak and strong wash steps are wellknown to those of skill in the art. For example, weak washes can be donewith standard buffers or culture media (e.g., phosphate buffered saline(buffer) DMEM (culture media), etc.).

E Culturing Under Internalizing Conditions.

As explained above, the cells are preferably cultured under“internalizing” conditions. Internalizing culture conditions areconditions in which the cell when bound by a member of a phage displaylibrary at an appropriate (e.g. internalizing) site or receptor,transports the bound member into the cell. This can involve transportinto a vesicle, into the endoplasmic reticulum, the golgi complex, orinto the cytosol of the cell itself.

Internalizing conditions are most easily achieved when the cells arecultured under conditions that mimic those of the cell in its nativestate. Thus many cells, e.g. epidermal cells, preferably grow adadherent layers attached to a basement membrane. Such cells moreeffectively internalize binding polypeptides and antibodies when theyare cultured as adherent monolayers. Chloroquine and serum free mediumboth avoid non specific internalization and enhance specificinternalization (ligands in the serum that induce the internalization ofreceptor of interest and take with them non specific phages being in theneighborhood). In addition, for internalization to occur, the cellsshould be cultured at a temperature and pH that permits internalization.Suitable temperature and pH range from about 35° C. to about 39° C. andfrom pH 6 to about pH 8, more preferably from about pH 6.5 to about pH7.5, with preferred temperature and pH being about 37° C. and pH 7.5respectively. In a preferred embodiment, the cells are preincubated inserum culture medium for about two hours before adding the phages andthe competitor (subtraction) cells.

F) Identification of Internalized Phase

The internalized phage display library members can be identifieddirectly or indirectly. Direct identification can be accomplished simplyby visualizing the phage within a cell e.g. via immunofluorescent orconfocal microscopy. Phage internalization can be identified by theirability to deliver a reporter gene that is expressed within the cell.The reporter gene can be one that produces a detectable signal (e.g. afluorescent (e.g. lux, green fluorescent protein, etc.) or colorimetricsignal (e.g. HRP, β-galactosidase) or can itself be a selectable marker(e.g. an antibiotic resistance gene). The use of both α-galactosidaseand GFP as reporter genes in such phage is described herein.

Alternatively, the phage display member can bear a marker (e.g. a label)and cells containing the internalized phage can be detected simply bydetection of the label (e.g. in a flow cytometer). The direct methodspreferably used for identification of the receptors or cells that arebound after selections are performed. It is noted that cell sortingapproaches (FACs) will work with identification of either surface boundor internalized phage. However, an additional level of specificity canbe achieved if the cells are first sorted for the presence ofinternalized phage prior to lysis. Direct methods are also used duringthe analysis phase to demonstrate that the phage selected are indeedinternalized.

Alternatively the internalized phage display library members can beidentified indirectly. In indirect detection methods the phage-displaylibrary member(s) do not need to be detected while they are presentwithin the cell. It is sufficient that they simply have beeninternalized.

Indirect identification is accomplished for example, by isolating andexpanding the phage that were internalized into the cells as describedbelow. Indirect identification is particularly well suited where theidentified phage display library members are going to be used insubsequent rounds of selection or to isolate bacteria harboringmonoclonal phage genomes for subsequent monoclonal phagecharacterization (that is for the analysis of selection results).

G) Isolation and Expansion of Internalized Phase.

It was a discovery of this invention that phage display library membersthat have been internalized into target cells (e.g. mammalian tumorcells) remain viable and can be recovered and expanded into a “selected”library suitable for subsequent rounds of selection and/or isolation andcharacterization of particular members.

As used herein, the term “recovery” is intended to include recovery ofthe infectious phage and/or recovery of the phage antibody gene and/orrecovery of a heterologous nucleic acid accompanying the antibody gene.

The internalized phage can be isolated and expanded using standardmethods. Typically these include lysing the cells (e.g., with 100 mMtriethylamine (high pH)), and using the lysate to infect a suitablebacterial host, e.g., E. coli TG1. The phage-containing bacteria arethen cultured according to standard methods (see, e.g., Sambrook supra.,Marks et al. (1991) J. Mol. Biol. 222: 581-597).

IV. Preparation and Modification of Internalizing Antibodies

As described below, once an internalizing antibody is identifiedadditional copies of the antibody can be prepared using either chemicalsynthetic means or by the use of recombinant expression systems. Inaddition, other “related” internalizing antibodies can be identified byscreening for antibodies that bind to the same epitope and/or bymodification of the identified internalizing antibody to producelibraries of modified antibody and then rescreening antibodies in thelibrary internalization.

A) Antibody Synthesis.

1) Chemical Synthesis.

The internalizing antibodies, once identified by the methods of thisinvention, can be chemically synthesized using well known methods ofpeptide synthesis. Solid phase synthesis in which the C-terminal aminoacid of the sequence is attached to an insoluble support followed bysequential addition of the remaining amino acids in the sequence onepreferred method for the chemical synthesis of single chain antibodies.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A., Merrifield et al. (1963) J. Am. Chem. Soc., 85:2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis, 2nded. Pierce Chem. Co., Rockford, Ill.

2) Recombinant Expression of Internalizing Antibodies.

In a preferred embodiment, the internalizing antibodies, once identifiedby the methods of this invention, are prepared using standard techniqueswell known to those of skill in the art. Nucleic acid sequences encodingthe internalizing antibodies are determined (e.g. via Sangersequencing). Using the sequence information, the nucleic acids may bechemically synthesized according to a number of standard methods knownto those of skill in the art. Oligonucleotide synthesis, is preferablycarried out on commercially available solid phase oligonucleotidesynthesis machines (Needham-VanDevanter et al. (1984) Nucleic Acids Res.12: 6159-6168) or manually synthesized using the solid phasephosphoramidite triester method described by Beaucage et. al. (Beaucageet. al. (1981) Tetrahedron Letts. 22(20): 1859-1862). Alternatively,nucleic acids encoding the antibody can be amplified and/or clonedaccording to standard methods.

Molecular cloning techniques to achieve these ends are known in the art.A wide variety of cloning and in vitro amplification methods suitablefor the construction of recombinant nucleic acids. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods ofproducing recombinant immunoglobulins are also known in the art. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Natl.Acad. Sci. USA 86: 10029-10033. In addition, detailed protocols for theisolation and cloning of the antibody are provided herein in theExamples, and in Schier et al. (1996) J. Mol. Biol., 263: 551-567.

B) Identification of Other Antibodies Binding the Same “Internalizing”Epitope.

Once one or more internalizing antibodies are identified by thescreening methods of this invention, other “related” internalizingantibodies can be identified by screening for antibodies thatcross-react with the identified internalizing antibodies, either at theepitope bound by the antibodies or with an idiotypic antibody raisedagainst the internalizing antibodies.

1) Cross-Reactivity with Anti-Idiotypic Antibodies.

The idiotype represents the highly variable antigen-binding site of anantibody and is itself immunogenic. During the generation of anantibody-mediated immune response, an individual will develop antibodiesto the antigen as well as anti-idiotype antibodies, whose immunogenicbinding site (idiotype) mimics the antigen.

Anti-idiotypic antibodies can be raised against the variable regions ofinternalizing antibodies identified in the screening systems of thisinvention using standard methods well known to those of skill in theart. Briefly, anti-idiotype antibodies can be made by injectinginternalizing antibodies of this invention, or fragments thereof (e.g.,CDRs)) into an animal thereby eliciting antiserum against variousantigenic determinants on the antibody, including determinants in theidiotypic region.

Methods for the production of anti-analyte antibodies are well known inthe art. Large molecular weight antigens (greater than approx. 5000Daltons) can be injected directly into animals, whereas small molecularweight compounds (less than approx. 5000 Daltons) are preferably coupledto a high molecular weight immunogenic carrier, usually a protein, torender them immunogenic. The antibodies produced in response toimmunization can be utilized as serum, ascites fluid, an immunoglobulin(Ig) fraction, an IgG fraction, or as affinity-purified monospecificmaterial.

Polyclonal anti-idiotype antibodies can be prepared by immunizing ananimal with the antibodies of this invention prepared as describedabove. In general, it is desirable to immunize an animal which isspecies and allotype-matched with the animal from which the antibody(e.g. phage-display library) was derived. This minimizes the productionof antibodies directed against non-idiotypic determinants. The antiserumso obtained is then usually absorbed extensively against normal serumfrom the same species from which the phage-display library was derived,thereby eliminating antibodies directed against non-idiotypicdeterminants. Absorption can be accomplished by passing antiserum over agel formed by crosslinking normal (nonimmune) serum proteins withglutaraldehyde. Antibodies with anti-idiotypic specificity will passdirectly through the gel, while those having specificity fornon-idiotypic determinants will bind to the gel. Immobilizing nonimmuneserum proteins on an insoluble polysaccharide support (e.g., sepharose)also provides a suitable matrix for absorption.

Monoclonal anti-idiotype antibodies can be produced using the method ofKohler et al. (1975) Nature 256: 495. In particular, monoclonalanti-idiotype antibodies can be prepared using hybridoma technologywhich comprises fusing (1) spleen cells from a mouse immunized with theantigen or hapten-carrier conjugate of interest (i.e., the antibodies orthis invention or subsequences thereof) to (2) a mouse myeloma cell linewhich has been selected for resistance to a drug (e.g., 8-azaguanine).In general, it is desirable to use a myeloma cell line which does notsecrete an immunoglobulin. Several such lines are known in the art. Apreferred cell line is P3X63Ag8.653. This cell line is on deposit at theAmerican Type Culture Collection as CRL-1580.

Fusion can be carried out in the presence of polyethylene glycolaccording to established methods (see, e.g., Monoclonal Antibodies, R.Kennett, J. McKearn & K. Bechtol, eds. N.Y., Plenum Press, 1980, andCurrent Topics in Microbiology & Immunology, Vol. 81, F. Melchers, M.Potter & N. L. Warner, eds., N.Y., Springer-Verlag, 1978). The resultantmixture of fused and unfused cells is plated out inhypoxanthine-aminopterin-thymidine (HAT) selective medium. Under theseconditions, only hybrid cells will grow.

When sufficient cell growth has occurred, (typically 10-14 dayspost-fusion), the culture medium is harvested and screened for thepresence of monoclonal idiotypic, anti-analyte antibody by any one of anumber of methods which include solid phase RIA and enzyme-linkedimmunosorbent assay: Cells from culture wells containing antibody of thedesired specificity are then expanded and recloned. Cells from thosecultures that remain positive for the antibody of interest are thenusually passed as ascites tumors in susceptible, histocompatible,pristane-primed mice.

Ascites fluid is harvested by tapping the peritoneal cavity, retestedfor antibody, and purified as described above. If a nonsecreting myelomaline is used in the fusion, affinity purification of the monoclonalantibody is not usually necessary since the antibody is alreadyhomogeneous with respect to its antigen-binding characteristics. Allthat is necessary is to isolate it from contaminating proteins inascites, i.e., to produce an immunoglobulin fraction.

Alternatively, the hybrid cell lines of interest can be grown inserum-free tissue culture and the antibody harvested from the culturemedium. In general, this is a less desirable method of obtaining largequantities of antibody because the yield is low. It is also possible topass the cells intravenously in mice and to harvest the antibody fromserum. This method is generally not preferred because of the smallquantity of serum which can be obtained per bleed and because of theneed for extensive purification from other serum components. However,some hybridomas will not grow as ascites tumors and therefore one ofthese alternative methods of obtaining antibody must be used.

2) Cross-Reactivity with the F5 or C1 Epitope.

Instead of the anti-idiotypic antibody, other internalizing antibodiescan be identified by cross-reactivity with the identified “prototypic”antibodies, against the epitope(s) used in the original selection.Competition between the “prototypic” internalizing antibodies and newcandidates in an epitope-mapping format establishes that the antibodiesare competing for the same epitope.

C) Phage Display Methods to Select Other “Related” InternalizingAntibodies.

1) Chain Shuffling Methods.

To create higher affinity antibodies, mutant scFv gene repertories,based on the sequence of a binding of an identified internalizingantibody, are created and expressed on the surface of phage. Higheraffinity scFvs are selected on antigen as described above and in theExamples.

One approach to creating modified single-chain antibody (scFv) generepertoires has been to replace the original V_(H) or V_(L) gene with arepertoire of V-genes to create new partners (chain shuffling) (Clacksonet al. (1991) Nature. 352: 624-628). Using chain shuffling and phagedisplay, the affinity of a human scFv antibody fragment which bound thehapten phenyloxazolone (phOx) was increased from 300 nM to 1 nM (300fold) (Marks et al. (1992) Bio/Technology 10: 779-783).

Thus, for example, to alter the affinity of an internalizing antibody, amutant scFv gene repertoire can be created containing the V_(H) gene ofthe internalizing antibody and a human V_(L) gene repertoire (lightchain shuffling). The scFv gene repertoire can be cloned into the phagedisplay vector pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res., 19:4133-4137) and after transformation a library of transformants isobtained.

Similarly, for heavy chain shuffling, the internalizing antibody V_(H)CDR1 and/or CDR2, and/or CDR3 and light chain are cloned into a vectorcontaining a human V_(H) gene repertoire to create a phage antibodylibrary transformants. For detailed descriptions of chain shuffling toincrease antibody affinity see Schier et al. (1996) J. Mol. Biol., 255:28-43, 1996.

2) Site-Directed Mutagenesis to Improve Binding Affinity.

The majority of antigen contacting amino acid side chains are located inthe complementarity determining regions (CDRs), three in the V_(H)(CDR1, CDR2, and CDR3) and three in the V_(L) (CDR1, CDR2, and CDR3)(Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothia et al.(1986) Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol., 217:133-151). These residues contribute the majority of binding energeticsresponsible for antibody affinity for antigen. In other molecules,mutating amino acids which contact ligand has been shown to be aneffective means of increasing the affinity of one protein molecule forits binding partner (Lowman et al. (1993) J. Mol. Biol., 234: 564-578;Wells (1990) Biochemistry, 29: 8509-8516). Site-directed mutagenesis ofCDRs and screening against c-erbB-2 may be used to generate C6antibodies having improved binding affinity and/or internalization of aknown internalizing antibody.

3) CDR Randomization to Produce Higher Affinity Human scFv.

In an extension of simple site-directed mutagenesis, mutant antibodylibraries can be created where partial or entire CDRs are randomized(V_(L) CDR1 and CDR2 and V_(H) CDR1, CDR2 and CDR3). In one embodiment,each CDR is randomized in a separate library, using the knowninternalizing antibody as a template. The CDR sequences of the highestaffinity mutants from each CDR library are combined to obtain anadditive increase in affinity. A similar approach has been used toincrease the affinity of human growth hormone (hGH) for the growthhormone receptor over 1500 fold from 3.4×₁₀ ⁻¹⁰ to 9.0×10⁻¹³ M (Lowmanet al. (1993) J. Mol. Biol., 234: 564-578).

V_(H) CDR3 often occupies the center of the binding pocket, and thusmutations in this region are likely to result in an increase in affinity(Clackson et al. (1995) Science, 267: 383-386). In one embodiment, fourV_(H) CDR3 residues are randomized at a time using the nucleotides NNS(see, e.g., Schier et al. (1996) Gene, 169: 147-155; Schier and Marks(1996) Human Antibodies and Hybridomas. 7: 97-105, 1996; and Schier etal. (1996) J. Mol. Biol. 263: 551-567, 1996).

4) Creation of Homodimers.

To create (scFv′)₂ antibodies, two internalizing scFvs are joined,either through a linker (e.g., a carbon linker, a peptide, etc.) orthrough a disulfide bond between, for example, two cysteins. Thus, forexample, to create disulfide linked scFv, a cysteine residue isintroduced by site directed mutagenesis between the myc tag and ahexahistidine tag at the carboxy-terminus of the antibodies describedherein. Introduction of the correct sequence can be verified by DNAsequencing. If the construct is in pUC119, the pelB leader directsexpressed scFv to the periplasm and cloning sites (NcoI and NotI) existto introduce F5 or C1 mutant scFv. The expressed scFv has the myc tag atthe C-terminus, followed by 2 glycines, a cysteine, and then 6histidines to facilitate purification by IMAC. After disulfide bondformation between the two cysteine residues, the two scFv are separatedfrom each other by about 26 amino acids (two 11 amino acid myc tags and4 glycines).

An scFv can be expressed from this construct, purified by IMAC, andanalyzed by gel filtration. To produce (scFv′)₂ dimers, the cysteine isreduced by incubation with 1 mM ∃-mercaptoethanol, and half of the scFvblocked by the addition of DTNB. Blocked and unblocked scFvs areincubated together to form (scFv′)₂ and the resulting material can beanalyzed by gel filtration. The affinity of the F5 and C1 scFv′ monomersand the F5 and C1 (scFv′)₂ dimers is determined by BIAcore.

In a particularly preferred embodiment, the (scFv′)₂ dimer is created byjoining the scFv′ fragments through a linker, more preferably through apeptide linker. This can be accomplished by a wide variety of means wellknown to those of skill in the art. For example, one preferred approachis described by Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448 (see also WO 94/13804).

5) Measurement of Antibody/Polypeptide Binding Affinity.

As explained above, selection for increased avidity involves measuringthe affinity of the antibody for the target antigen (e.g., c-erbB-2).Methods of making such measurements are described in detail in copendingapplication U.S. Ser. No. 08/665,202. Briefly, for example, the K_(d) ofF5, C1, or an F5- or C1-derived antibody the kinetics of binding toc-erbB-2 are determined in a BIAcore, a biosensor based on surfaceplasmon resonance. For this technique, antigen is coupled to aderivatized sensor chip capable of detecting changes in mass. Whenantibody is passed over the sensor chip, antibody binds to the antigenresulting in an increase in mass that is quantifiable. Measurement ofthe rate of association as a function of antibody concentration can beused to calculate the association rate constant (k_(on)). After theassociation phase, buffer is passed over the chip and the rate ofdissociation of antibody (k_(off)) determined. K_(on) is typicallymeasured in the range 1.0×10² to 5.0×10⁶ and k_(off) in the range1.0×10⁻¹ to 1.0×10⁻⁶. The equilibrium constant K_(d) is often calculatedas k_(off)/k_(on) and thus is typically measured in the range 10⁻⁵ to10-12. Affinities measured in this manner correlate well with affinitiesmeasured in solution by fluorescence quench titration.

V. Libraries and Vectors

In another embodiment, this invention provides libraries and vectors forpractice of the methods described herein. The libraries are preferablypolyvalent libraries, including diabody libraries and more preferablyincluding multi-valent single chain antibody libraries (e.g. scFv),(e.g., expressed by phage).

The libraries can take a number of forms. Thus, in one embodiment thelibrary is a collection of cells containing members of the phage displaylibrary, while in another embodiment, the library consists of acollection of isolated phage, and in still library consists of a libraryof nucleic acids encoding a polyvalent phage display library. Thenucleic acids can be phagemid vectors encoding the antibodies and readyfor subcloning into a phage vector or the nucleic acids can be acollection of phagemid already carrying the subcloned antibody-encodingnucleic acids.

VI. Kits For Selecting Internalizing Antibodies

In another embodiment, this invention provides kits for practice of themethods described herein. The kits preferably include members of a phagedisplay library (e.g., as phage particles, as vectors, or as cellscontaining phage). The assay kits can additionally include any of theother components described herein for the practice of the assays of thisinvention. Such materials preferably include, but are not limited to,helper phage, one or more bacterial or mammalian cell lines, buffers,antibiotics, labels, and the like.

In addition the kits may optionally include instructional materialscontaining directions (i.e., protocols) disclosing the selection methodsdescribed herein. While the instructional materials typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Creation of a Non-Immune Human Fab Phase Antibody LibraryContaining 10⁹-10¹¹ members

Manipulation of previous 10⁷ member phage display libraries revealed twomajor limitations: 1) expression levels of Fabs was too low to produceadequate material for characterization, and 2) the library wasrelatively unstable. These limitations are a result of creating thelibrary in a phage vector, and the use of the cre-lox recombinationsystem. We therefore decided that the best approach for this project wasto create a very large scFv library using a phagemid vector. The goalwas to produce a library at least 100 times larger than our previous3.0×10⁷ member scFv library. The approach taken was to clone the V_(H)and V_(L) library on separate replicons, combine them into an scFv generepertoire by splicing by overlap extension, and clone the scFv generepertoire into the phage display vector pHEN1. Human peripheral bloodlymphocyte and spleen RNA was primed with IgM heavy chain constantregion and, kappa and lambda light chain constant region primers andfirst strand cDNA synthesized. 1st strand cDNA was used as a templatefor PCR amplification of VH Vκk and Vλgene repertoires.

The V_(H) gene repertoires were cloned into the vector pUC119Sfi-Not asNco1-NotI fragments, to create a library of 8.0×10⁸ members. The librarywas diverse by PCR fingerprinting. Single chain linker DNA was splicedonto the V_(L) gene repertoires using PCR and the repertoire cloned asan XhoI-NotI fragment into the vector pHENIXscFv to create a library of7.2×10⁶ members. The V_(H) and V_(L) gene repertoires were amplifiedfrom their respective vectors and spliced together using PCR to createan scFv gene repertoire. The scFv gene repertoire was cloned as anNcoI-NotI fragment into the vector to create an scFv phage antibodylibrary of 7.0×10⁹ members. The library was diverse as determined byBstN1 fingerprinting.

To verify the quality of the library, phage were prepared and selectedon 14 different protein antigens. The results are shown in Table 1. scFvantibodies were obtained against all antigens used for selection, withbetween 3 and 15 unique scFv isolated per

TABLE 1 Results of phage antibody library selections. Number ofPercentage (number) different of ELISA positive antibodies Proteinantigen used for selection clones isolated FGF Receptor ECD 69 (18/26)15 BMP Receptor Type I ECD 50 (12/24) 12 Activin Receptor Type I ECD 66(16/24) 7 Activin Receptor Type II ECD 66 (16/24) 4 Erb-B2 ECD 91(31/34) 14 VEGF 50 (48/96) 6 BoNT/A 28 (26/92) 14 BoNT-A C-fragment 95(87/92) 10 BoNT/B 10 (9/92)  5 BoNT/C 12 (11/92) 5 BoNT/E 9 (8/92) 3Bungarotoxin 67 (64/96) 15 Cytochrome b5 55 (53/96) 5 Chlamydiatrachomatis EB 66 (63/96) 7 For each antigen (column 1), the number andthe percentage of positive clones selected (column 2) and the number ofdifferent antibodies isolated (column 3) is indicatedantigen (average 8.7) (Table 1). This compares favorably to resultsobtained from smaller scFv libraries (1 to a few binders obtainedagainst only 70% of antigens used for selection). Affinities of 4anti-ErbB-2 scFv and 4 anti-Botulinum scFv were measured using surfaceplasmon resonance in a BIAcore and found to range from 4.0×10⁻⁹ M to2.2×10⁻¹⁰ M for the anti-ErbB2 scFv and 2.6×10⁻⁸ M to 7.15×10⁻⁸ M forthe anti-Botulinum scFv (Table 2). scFv were highly specific for theantigen used for selection (FIG. 2). The library could also besuccessfully selected on complex mixtures of antigen.

TABLE 2. Affinities and binding kinetics of anti-BoNT A C-fragment andanti-Erb-B2 scFv. Specificity and clone K_(d) (×10⁻⁹M) k_(on)(×10⁵M⁻¹s⁻¹) k_(off)(×10⁻³s⁻¹) ErbB-2 B7A 0.22 4.42 0.1 ErbB-2 G11D 0.482.19 0.11 ErbB-2 A11A 0.49 3.69 0.18 ErbB-2 F5A 4.03 1.62 0.65 BoNT-A2A9 26.1 0.25 0.66 BoNT-A 2H6 38.6 2.2 8.5 BoNT-A 3F6 66.0 4.7 30.9BoNT-A 2B6 71.5 1.1 7.8 Association (k_(on)) and dissociation (k_(off))rate constants for purified scFvs were measured using surface plasmonresonance (BIAcore) and K_(d) calculated as (k_(off)/k_(on)).

For example, selection on Chlamydia trachomatis elementary bodies (thecausative organism of Chlamydial disease) yielded seven thatspecifically recognized chlamydia (Table 1). The scFv could besuccessfully used in a number of immunologic assays including ELISA,immunofluorescence, Western blotting, epitope mapping andimmunoprecipitation. The number of binding antibodies for each antigen,and the affinities of the binding scFv are comparable to resultsobtained from the best phage antibody libraries (Table 3). Thus thelibrary was established as a source of panels of human antibodiesagainst any antigen with affinities at least equivalent to the secondarymurine response.

TABLE 3 Comparison of protein binding antibodies selected fromnon-immune phage-display antibody libraries. Number Average number Rangeof of protein of antibodies Number affinities for antigens per proteinof affinities protein antigens Library Library size and type* studiedantigen measured K_(d) (×10⁻⁹M) Marks et al (1991) J. 3.0 × 10⁷ (scFv,N) 2 2.5 1 100-2000 Mol. Biol. 222: 581-597 Nissim et al (1994) 1.0 ×10⁸ (scFV, SS) 15 2.6 ND ND EMBO J. 13: 692-698 DeKruif et al (1995) J.3.6 × 10⁸ (scFv, SS) 12 1.9 3 100-2500 Mol. Biol. 248: 97-105 Griffithset al (1994) 6.5 × 10¹⁰ (Fab, SS) 30 4.8 3 7-58 EMBO J. 13: 3245-3260Vaughan et al (1996) 1.4 × 10¹⁰ (scFv, N) 3 7.0 3 4.2-8.0  NatureBiotechnology. 14: 309-314 Present Examples 6.7 × 10⁹ (scFv, N) 14 8.7 80.22-71.5  *For library type, N = V-gene repertoires obtained fromV-genes rearranged in vivo; SS = semi-synthetic V-genes constructed fromcloned V-gene segments and synthetic oligonucleotides encoding V_(H)CDR3. ND = not determined.

These experiments demonstrate the creation of a high complexity humanscFv phage antibody library from which a panel of high affinity humanscFv can be generated against any purified antigen. Such a library isideal for probing the surface of cells to identify novel cell surfacemarkers.

Example 2 Uptake of scFV into Cells by Receptor Mediated Endocytosis andSubsequent Recovery

The 7.0×10⁹ member scFv phage antibody library described above wasselected on the malignant breast tumor cell lines MB231 and ZR-75-1,both with and without negative selections on the normal breast cell lineHBL100, Similar results were obtained as described in section above.scFv were isolated that could not distinguish malignant fromnon-malignant cell lines.

To increase the specificity of selections, it was hypothesized thatphage binding cell surface receptors could be taken up into cells byreceptor mediated endocytosis and could then be recovered from cells bylysing the cells. This assumed: 1) that phage could be internalized byreceptor mediated endocytosis and 2) that phage could be recovered inthe infectious state from within cells prior to lysosomal degradation.The ability to select for internalized phage antibodies would have twomajor benefits: 1) the identification of antibodies that bind toreceptors capable of internalization and 2) an added level ofspecificity in the selection process. Identification of antibodies whichare internalized would be highly useful for many targeted therapeuticapproaches where internalization is essential (e.g. immunotoxins,targeted liposomes, targeted gene therapy vectors and others).

A) Receptor Mediated Internalization of F5 or C1 Phase

To determine proof of principle, we utilized C6.5 phage and C6.5 diabodyphage (see, copending application U.S. Ser. No. 08/665,202). We havepreviously shown that C6.5 scFv is internalized, but at a slow rate, andthat the C6.5 diabody is somewhat better internalized (probably becauseit causes receptor dimerization). C6.5 phage, C6.5 diabody phage or anirrelevant anti-Botulinum phage were incubated with SKBR3 cells (ErbB2expressing breast tumor cell line) at either 37° C. or 4° C. andnon-internalized phage removed by sequential washing with PBS and low pHglycine buffer. The cells were then permeabilized and biotinylatedanti-M13-antibody added followed by streptavidin Texas Red. Cells werethen examined by using a confocal microscope. Both C6.5 phage and C6.5diabody phage were observed within the cytoplasm). Approximately 1% ofcells had internalized C6.5 phage and 20% of the cells had internalizedC6.5 diabody phage. There was no internalization of the anti-Botulinumphage.

To determine if infectious phage could be specifically taken up andrecovered from within cells, C6.5 phage or C6.5 diabody phage wereincubated with SKBR3 cells at 37° C. Non bound phage were removed bywashing with PBS and phage bound to the cell surface were eluted bywashing twice with low pH glycine. The cells were then lysed and eachfraction (the first and second glycine washes and the cytoplasmicfraction) used to infect E. coli TG1. Twenty times (C6.5) or 30 times(C6.5 diabody) more phage were bound to the cell surface than theanti-Botulinum phage (glycine 1 wash) (Table 4). After the secondglycine wash, the titre of infectious phage from the cell surfacedecreased, indicating that washing was effective at removing surfacebound phage (Table 4). After cell lysis, the titer increased more than10 fold (C6.5 phage) or 50 fold (C6.5 diabody phage) from the secondglycine wash. We believe this titre represents phage recovered frominside the cell. Recovery of phage from inside the cell was 100 timeshigher for ErbB2 binding C6.5 than for anti-Botulinum phage and 200 foldhigher for C6.5 diabody phage (Table 4).

TABLE 4 Titer of cell surface bound phage and internalized phage. Lysedcell Phage specificity 1st glycine wash 2nd glycine wash fractionanti-Botulinum 6.0 × 10⁵ 1.0 × 10⁵ 6.0 × 10⁵ Anti-ErbB2 1.2 × 10⁷ 5.2 ×10⁶ 6.8 × 10⁷ (C6.5 scFv) Anti-ErbB2 1.8 × 10⁷ 2.8 × 10⁶ 1.7 × 10⁷ (C6.5diabody) 5.0 × 10¹¹ phage (anti-Botulinum or anti-ErbB2) were incubatedwith approximately 1.0 × 10⁵ ErbB2 expressing SKBR3 cells at 37° C.Cells were washed 10 times with PBS and surface bound phage eluted withtwo low pH glycine washes. The cells were then washed once with PBS andthe cells lysed to release internalized phage. The phage titer was thendetermined for each of the glycine washes and for the lysed cellfraction by infection of E. coli TG1.

Taken together, the results indicate that: 1) phage binding cell surfacereceptors can be taken up by cells and the infectious phage recoveredfrom the cytoplasm. The amount of uptake is significantly greater thanuptake of non-binding phage, and the 100 to 200 fold difference is wellwithin the range that would allow enrichment from a library. What isunknown from the results is whether the phage antibodies are mediatingreceptor mediated internalization or whether they are merely taken upafter binding by membrane turnover.

B) Selection and Characterization of Internalizing Antibodies from aPhase Antibody Library

The results described above encouraged us to attempt selection of thephage antibody library described above to identify new phage antibodiesthat were internalized. Phage antibodies were rescued from the libraryand selected on SKBR3 cells. For selection, phage were incubated withcells at 37° C., non-binding phage removed by washing cells with PBS andphage bound to cell surface antigens removed by sequential washes withlow pH glycine. Cells were then lysed to release internalized phage andthe lysate used to infect E. Coli TG1 to prepare phage for the nextround of selection. Three rounds of selection were performed. Onehundred clones from each round of selection were analyzed for binding toSKBR3 cells and to ErbB2 extracellular domain by ELISA. We hypothesizedthat we were likely to obtain binders to ErbB2 since SKBR3 cells areknown to express high levels and ErbB2 is a receptor which is known tobe internalized. After each round of selection, the titer of phagerecovered from the cytoplasm increased (Table 5). After the third round,45% of the clones were positive SKBR3 cell binding and 17% bound ErbB2(Table 5).

TABLE 5 Results of selection of a phage antibody library forinternalization. Round of # of phage in # of cells # of % SKBR3 % ErbB2selection cell lysate lysed phage/cell binders binders 1 3.5 × 10⁴ 2.8 ×10⁶ 0.013 ND ND 2 1.2 × 10⁵ 2.8 × 10⁶ 0.038 ND ND 3 7.5 × 10⁶ 2.8 × 10⁶3.75 45% 17% For each round of selection, the titer of phage in lysedcells, number of cells lysed and number of phage per cell is indicated.After the third round, individual clones were analyzed for binding toSKBR3 cells by ELISA and to ErbB2 ECD by ELISA.

To estimate the number of unique binders, the scFv gene from ELISApositive clones was PCR amplified and fingerprinted by digestion withBstN1. Two unique restriction patterns were identified. The scFv geneswere sequenced and 2 unique ErbB2 binding scFv identified. Similaranalysis of SKBR3ELISA positive clones that did not bind ErbB2identified an additional 11 unique scFv.

To verify that phage antibodies were specific for SKBR3 cells, phagewere prepared from each unique clone and analyzed for binding to SKBR3cells (high ErbB2 expression) as well as 2 other epithelial tumor celllines (SK-OV-3, moderate ErbB2 expression and MCF7, low ErbB2expression) and a normal breast cell line (HS578B). Each unique clonespecifically stained tumor cell lines but not the normal breast cellline.

SKBR3 and MCF7 cells were incubated with phage antibodies C6.5 (positivecontrol), 3TF5 and 3 GH7. The latter two clones were isolated from thelibrary, with 3TF5 binding ErbB2 and the antigen bound by 3 GH7 unknown.All 3 phage antibodies intensely stain SKBR3 cells (the selecting cellline and high ErbB2 expresser. C6.5 phage weakly stain MCF7 cells (lowErbB2 expressor). The anti-ErbB2 clone 3TF5 from the library stains MCF7cells much more intensely then C6.5, as does 3 GH7.

SKBR3, SK-OV-3, MCF7 and HST578 cells were studied using native purifiedscFv 3TF5 and 3 GH7. For these studies, the scFv genes were subclonedinto a vector which fuses a hexahistidine tag to the scFv C-terminus.scFv was then expressed, harvested from the bacterial periplasm andpurified by immobilized metal affinity chromatography. The two scFvintensely stain SKBR3 cells, and do not stain the normal breast cellline HST578. There is minimal staining of the low ErbB2 expressing cellline MCF7 and intermediate staining of SK-OV-3 cells (moderate ErbB2expresser). In general, the intensity of staining is less than seen withphage. This is to be expected since the secondary antibody for phagestaining recognizes the major coat protein (2500 copies/phage) resultingin tremendous signal amplification.

The anti-ErbB2 phage antibody 3TF5 was studied further to determine ifit was indeed internalized. This antibody was selected for initial studysince its internalization could be compared to ErbB2 binding C6.5.5.0×10¹¹ 3TF5 or C6.5 phage were incubated with SKBR3 cells at 37° C. orat 4° C. After washing with PBS, 3TF5 phage stained cells more intenselythan C6.5 phage. After washing with low pH glycine, confocal microscopyrevealed that 3TF5 phage were internalized by greater than 95% of cells,while C6.5 was internalized by only a few percent of cells. Incubationof either antibody at 4° C. led to no internalization.

The native purified 3TF5 scFv was similarly analyzed and was alsoefficiently internalized by SKBR3 cells. It should be noted that thenative 3TF5 scFv existed only as a monomer with no appreciabledimerization or aggregation as determined by gel filtration.

These experiments demonstrate that phage antibodies can be internalizedby cells and recovered from the cytoplasm. Phage that bind aninternalizing cell surface receptor can be enriched more than 100 foldover non-binding phage. This level of enrichment is greater than thatachieved by selecting on the cell surface. We have applied this approachto library selection and isolated phage antibodies that bind and areinternalized by SKBR-3 cells. Several of these antibodies bind to ErbB2,but are more efficiently internalized than antibodies such as C6.5 thatwere generated by selecting on pure antigen. Many other antibodies havebeen isolated that bind specifically to SKBR-3 and other breast tumorcell lines and are efficiently internalized. These antibodies shouldprove useful for tumor targeting and for identifying potentially novelinternalizing tumor cell receptors.

Example 3 Increasing the Affinity of Antibody Fragments with the DesiredBinding Characteristics by Creating Mutant Phase Antibody Libraries andSelecting on the Appropriate Breast Tumor Cell Line

Phage display has the potential to produce antibodies with affinitiesthat cannot be produced using conventional hybridoma technology. Ultrahigh affinity human antibody fragments could result in excellent tumorpenetration, prolonged tumor retention, and rapid clearance from thecirculation, leading to high specificity. We therefore undertook aseries of experiments to develop methodologies to generate ultra highaffinity human antibody fragments. Experiments were performed to answerthe following questions: 1) What is the most effective way to select andscreen for rare higher affinity phage antibodies amidst a background oflower affinity binders; 2 What is the most effective means to removebound phage from antigen, to ensure selection of the highest affinityphage antibodies; 3) What is the most efficient techniques for makingmutant phage antibody libraries (random mutagenesis or site directedmutagenesis; 4) What region of the antibody molecule should be selectedfor mutagenesis to most efficiently increase antibody fragment affinity.

To answer these questions, we studied the human scFv C6.5, which bindsthe extracellular domain (ECD) of the tumor antigen ErbB-2 (32) with aK_(d) of 1.6×10⁻⁸ M and k_(off) of 6.3×10⁻³ s⁻¹ (Schier et al. (1995)Immunotechnology, 1: 63-71). Isolation and characterization of C6.5 isdescribed briefly below and in detail in copending application U.S. Ser.No. 08/665,202).

Despite excellent tumor:normal tissue ratios in vivo, quantitativedelivery of C6.5 was not adequate to cure tumors in animals usingradioimmunotherapy (Schier et al. (1995) Immunotechnology, 1: 63-71). Toimprove the quantitative delivery of antibody to tumor, the affinity ofC6.5 was increased. First, techniques were developed that allowedselection of phage antibodies on the basis of affinity, rather thandifferential growth in E. coli or host strain toxicity (Schier et al.(1996) J. Mol. Biol. 255: 28-43; Schier et al. (1996) Gene 169: 147-155;Schier et al. (1996) Human antibodies and hybridomas 7: 97-105). Next,we determined which locations in the scFv gene to mutate to achieve thegreatest increments in affinity (Schier et al. (1996) J. Mol. Biol. 255:28-43; Schier et al. (1996) Gene; Schier et al. (1996) J. Mol. Biol.263: 551-567). Random mutagenesis did not yield as great an increment inaffinity as site directed mutagenesis of the complementarity determiningregions (CDRs) that contain the amino acids which contact antigen.Results from diversifying the CDRs indicated that: 1) the greatestincrement in affinity was achieved by mutating the CDRs located in thecenter of the binding pocket (V_(L) and V_(H) CDR3); 2) half of the CDRresidues have a structural role in the scFv and when mutated return aswild-type; and 3) these structural residues can be identified prior tolibrary construction by modeling on a homologous atomic crystalstructure. These observations led to development of a generic strategyfor increasing antibody affinity where mutations are randomly introducedsequentially into V_(H) and V_(L) CDR3, with conservation of residuespostulated to have a structural role by homology modeling (Schier et al.(1996) J. Mol. Biol. 263: 551-567). Using this approach, the affinity ofC6.5 was increased 1200 fold to a K_(d) of 1.3×10⁻¹¹ M (Id.).

Biodistribution studies revealed a close correlation between affinityand the percent injected dose of scFv/gram of tumor (% ID/g) at 24 hours(Adams et al. (1998) Cancer Res. 58: 485-490). The greatest degree oftumor retention was observed with ¹²⁵I-C6ML3-9 (1.42% ID/g,K_(d)=1.0×10⁻⁹ M). Significantly less tumor retention was achieved with¹²⁵I-C6.5 (0.80% ID/g, K_(d)=1.6×10⁻⁸) and C6G98A (0.19% ID/g,K_(d)=3.2×10⁻⁷ M). The tumor:normal organ ratios also reflected thedifferences in affinity, e.g. tumor:blood ratios of 17.2, 13.3, 3.5 and2.6, and tumor to liver ratios of 26.2, 19.8, 4.0 and 3.1 for C6ML3-9,C6.5 and C6G98A respectively at 24 hours. Studies of the higher affinityscFv are pending. The results demonstrate our ability to increaseantibody affinity to values not achievable from hybridoma technology andconfirm the importance of affinity in tumor targeting

Example 4 Preclinical Development of C6.5 Based Breast Cancer Therapies

Two approaches have been collaboratively pursued to develop C6.5 basedbreast cancer therapies. C6.5 based molecules are being engineered forradioimmunotherapy. To increase quantitative tumor delivery andretention of antibody fragment, dimeric scFv ‘diabodies’ were created byshortening the linker between the V_(H) and V_(L) domains from 15 to 5amino acids. Consequently, pairing occurs between complementary domainsof two different chains, creating a stable noncovalently bound dimerwith two binding sites. In vitro, diabodies produced from the V-genes ofC6.5 have a significantly higher apparent affinity and longer retentionon the surface of SK-OV-3 cells compared to C6.5 scFv (T_(1/2)>5 hr vs.5 min) (Adams et al. (1998) Brit. J. Cancer.). Biodistribution studiesof C6.5 diabody revealed 6.5% ID/g tumor at 24 hours compared to only 1%ID/g for C6.5 scFv. When diabody retentions were examined over 72 hoursand cumulative area under the curve (AUC) values determined, theresulting tumor:organ AUC ratios were greater than reported for othermonovalent or divalent scFv molecules. The therapeutic potential ofthese molecules is being examined in radioimmunotherapy studies in nudemice. Since in vivo characterization of c6.5 based molecules was notformally one of the technical objectives, we are continuing to use theaffinity mutants of C6.5 and C6.5 based diabodies to study therelationship between antibody affinity, size and valency and specifictumor targeting as part of NIH R01 CA65559-01A1.

In another collaboration C6.5 based molecules are being used to targetdoxorubicin containing stealth liposomes to ErbB2 expressing breastcancers (Kirpotin et al. (1997) Biochemistry. 36: 66-75). To facilitatechemical coupling of the scFv to liposomes, the C6.5 gene was subclonedinto an E. coli expression vector resulting in addition of a freecysteine residue at the C-terminus of the scFv. Purified C6.5cys scFvwas conjugated to liposomes and in vitro uptake determined using SKBR3cells. Total uptake was 3.4 mmol phospholipid/10⁶ cells at 6 hour, with70% of the uptake internalized. The uptake is comparable to thatachieved using the 4D5 anti-HER2Fab′ from Genentech. There was no uptakeof unconjugated liposomes. The results indicate that C6.5 binds to aErbB2 epitope that results in internalization at a rate comparable tothe best internalizing antibody produced from hybridomas (4D5). In vivotherapy studies in scid mice indicated that C6.5 targeted liposomescaused a greater degree of tumor regression and a higher cure rate thanuntargeted liposomes or a combination of untargeted liposomes andsystemic 4D5 antibody.

Conclusions

The experiments described herein establish that A large (7.0×10⁹ member)phage antibody library has been created which can provide panels ofhuman antibodies to purified antigens with affinities comparable to theaffinities of antibodies produced by murine immunization. The phageantibodies binding cell surface receptors can be can be internalized bycells and recovered in an infectious state from within the cell.Methodologies were developed which permit enrichment of internalizingphage antibodies over non-internalizing antibodies more than 100 fold.These methodologies were then applied to select new scFv antibodies thatbind to internalizing receptors on SKBR-3 cells. Several of theseantibodies bind to ErbB2, but are internalized more efficiently thanC6.5 based scFv. Many more antibodies bind to unknown internalizingreceptors. All of these scFv bind specifically to SKBR-3 cells orrelated tumor cell lines. The results indicate that this selectionapproach is a powerful approach to generate antibodies that candistinguish one cell type (malignant) from another (non-malignant).Moreover, we have demonstrated that it is not only possible to selectfor binding, but to select for function (internalization). In the nearterm, we will further characterize the antibodies isolated with respectto specificity, and in the case of ErbB2 binding scFv, affinity. In thelonger term we will use these reagents to: 1) study the effect ofaffinity and valency on the rate of internalization; and 2) identify theantigens bound using immunoprecipitation. It is likely that the resultswill lead to the identification of novel internalizing tumor cellsurface receptors which will be useful therapeutic targets. If thisapproach proves useful, we plan on applying it to primary tumor cellsand DCIS. We also intend to evaluate 3TF5 (ErbB2 binding scFv which isinternalized faster than C6.5) for liposome targeting. It is possiblethat it will be more effective than C6.5

In addition, the experiments demonstrate that methodologies forincreasing antibody affinity in vitro to values not previously achievedin vivo. We have applied these methodologies to generate novel ErbB2binding scFv.

Example 5 Selection of Internalizing Antibodies from Phase Libraries

In this example, we studied a human scFv (C6.5) that binds ErbB2 todetermine the feasibility of directly selecting internalizing antibodiesfrom phage libraries and to identify the most efficient display format.Using wild type C6.5 scFv displayed monovalently on a phagemid, wedemonstrate that anti-ErbB2 phage antibodies can undergo receptormediated endocytosis. Using affinity mutants and dimeric diabodies ofC6.5 displayed as either single copies on a phagemid or multiple copieson phage, we define the role of affinity, valency, and display format onphage endocytosis and identify the factors that lead to the greatestenrichment for internalization. Phage displaying bivalent diabodies ormultiple copies of scFv were more efficiently endocytosed than phagedisplaying monomeric scFv and recovery of infectious phage was increasedby preincubation of cells with chloroquine. Measurement of phagerecovery from within the cytosol as a function of applied phage titerindicates that it is possible to select for endocytosable antibodies,even at the low concentrations that would exist for a single phageantibody member in a library of 10⁹.

A) Material and Methods

1) Cells

The SKBR3 breast tumor cell line was obtained from ATCC and grown inRPMI media supplemented with 10% FCS (Hyclone) in 5% CO₂ at 37° C.

2) Antibodies and Antibody Phase Preparations

The C6.5 scFv phage vector was constructed by subcloning the C6.5 geneas a Sfi I/Not I fragment from scFv C6.5 pHEN1 (Schier et al. (1995)Immunotechnology 1: 63-71) into the phage vector fd/Sfi I/Not I (a giftof Andrew Griffiths, MRC Cambridge, UK). The C6.5 diabody phagemidvector was constructed by subcloning the C6.5 diabody gene (Adams et al.(1998) Brit. J. Cancer. 77: 1405-1412, 1998) as a NcoI/NotI fragmentinto pHEN1 (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137).The anti-botulinum scFv phagemid (clone 3D12) (Amersdorfer et al. (1997)Infection and Immunity. 65: 3743-3752) C6.5 scFv phagemid (Schier et al.(1995) Immunotechnology 1: 63-71) and scFv C6ML3-9 scFv phagemid (Schieret al. (1996) J. Mol. Biol. 263: 551-567) in pHEN1 have been previouslydescribed. Phage were prepared (Sambrook et al. (1990). Molecularcloning—a laboratory manual., New York: Cold Spring Harbor Laboratory)from the appropriate vectors and titered on E. coli TG1 as previouslydescribed (Marks et al. (1991) J. Mol. Biol. 222: 581-597) usingampicillin (100 μg/ml) resistance for titration of constructs in pHEN1and tetracyline (50 μg/ml) for titration of constructs in fd. SolubleC6.5 scFv, C6.5 diabody and anti-botulinum scFv were expressed from thevector pUC119mycHis (Schier et al. (1995) Immunotechnology 1: 63-71) andpurified by immobilized metal affinity chromatography as describedelsewhere (Id.)).

3) Detection of Internalized Native Antibody Fragments and PhaseAntibodies

SKBR3 cells were grown on coverslips in 6-well culture plates (Falcon)to 50% of confluency. Culture medium was renewed 2 hours prior to theaddition of 5.10¹¹ cfu/ml of phage preparation (the phage preparationrepresenting a maximum of 1/10 of the culture medium volume) or 20 μg/mlof purified scFv or diabody in phosphate buffered saline, pH 7.4 (PBS).After 2 hours of incubation at 37° C., the wells were quickly washed 6times with ice cold PBS and 3 times for 10 minutes each with 4 mL ofstripping buffer (50 mM glycine pH 2.8, 0.5 M NaCl, 2M urea, 2%polyvinylpyrrolidone) at RT. After 2 additional PBS washes, the cellswere fixed in 4% paraformaldehyde (10 minutes at RT), washed with PBS,permeabilized with acetone at −20° C. (30 seconds) and washed again withPBS. The coverslips were saturated with PBS-1% BSA (20 min. at RT).Phage particles were detected with biotinylated anti-M13 immunoglobulins(5 Prime-3 Prime, Inc, diluted 300 times) (45 min. at RT) and Texasred-conjugated streptavidin (Amersham, diluted 300 times) (20 min. atRT). Soluble scFv and diabodies containing a C-terminal myc peptide tagwere detected with the mouse mAb 9E10 (Santa Cruz Biotech, diluted 100times) (45 min. at RT), anti-mouse biotinylated immunoglobulins(Amersham, diluted 100 times) and Texas red-conjugated streptavidin.Optical confocal sections were taken using a Bio-Rad® MRC 1024 scanninglaser confocal microscope. Alternatively, slides were analyzed with aZeiss Axioskop UV fluorescent microscope.

4) Recovery and Titration of Cell Surface Bound or Internalized Phase

Subconfluent SKBR3 cells were grown in 6-well plates. Culture medium wasrenewed 2 hours prior to the experiment. Cells were incubated forvarying times with different concentrations of phage preparation at 37°C. Following PBS and stripping buffer washes, performed exactly asdescribed above for detection of internalized native antibody fragmentsand phage antibodies, the cells were washed again twice with PBS andlysed with 1 mL of 100 mM triethylamine (TEA). The stripping bufferwashes and the TEA lysate were neutralized with ½ volume of Tris-HCl 1M,pH 7.4. For some experiments, cells were trypsinized after the threestripping buffer washes, collected in a 15 ml Falcon tube, washed twicewith PBS and then lysed with TEA. In experiments performed in thepresence of chloroquine, SKBR3 cells were preincubated for two hours inthe presence of complete medium containing 50 μM chloroquine prior tothe addition of phage. Corresponding control samples in the absence ofchloroquine were prepared at the same time. For all experiments, phagewere titered on E. coli TG1 as described above.

B) Results

1) The Model System Utilized to Study Phase Antibody Internalization

The human anti-ErbB2 scFv C6.5 was obtained by selecting a human scFvphage antibody library on recombinant ErbB2 extracellular domain (13).C6.5 scFv binds ErbB2 with a K_(d)=1.6×10⁻⁸ M and is a stable monomericscFv in solution with no tendency to spontaneously dimerize or aggregate(Schier et al. (1995) Immunotechnology 1: 63-71). To determine theimpact of affinity on internalization, we studied a scFv (C6ML3-9) whichdiffers from C6.5 by 3 amino acids (Schier et al. (1996) J. Mol. Biol.263: 551-567). C6ML3-9 scFv is also a stable monomer in solution andbinds the same epitope as C6.5 scFv but with a 16 fold lower K_(d)(1.0×10⁻⁹ M) (Schier et al. (1996) J. Mol. Biol. 263: 551-567; Adams etal. (1998) Cancer Res. 58: 485-490). Since receptor homodimerizationappears to typically be requisite for antibody internalization we alsostudied the dimeric C6.5 diabody (Adams et al. (1998) Brit. J. Cancer.77: 1405-1412, 1998). Diabodies are scFv dimers where each chainconsists of heavy (V_(H)) and light (V_(L)) chain variable domainsconnected using a peptide linker which is too short to permit pairingbetween domains on the same chain. Consequently, pairing occurs betweencomplementary domains of two different chains, creating a stablenoncovalent dimer with two binding sites (Holliger et al. (1993) Proc.Natl. Acad. Sci. 90: 6444-6448). The C6.5 diabody was constructed byshortening the peptide linker between the Ig V_(H) and V_(L) domainsfrom 15 to 5 amino acids and binds ErbB2 on SKBR3 cells bivalently witha K_(d) approximately 40 fold lower than C6.5 (4.0×10⁻¹⁰ M) (Adams etal. (1998) Brit. J. Cancer. 77: 1405-1412, 1998).

Native C6.5 scFv and C6.5 diabody was expressed and purified from E.coli and analyzed for endocytosis into ErbB2 expressing SKBR3 breasttumor cells by immunofluorescent confocal microscopy. As expected,monomeric C6.5 scFv is not significantly internalized whereas thedimeric C6.5 diabody can be detected in the cytoplasm of all cellsvisualized.

For subsequent experiments, the C6.5 and C6ML3-9 scFv and C6.5 diabodygenes were subcloned for expression as pill fusions in the phagemidpHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137). Thisshould yield phagemid predominantly expressing a single scFv ordiabody-pIII fusion after rescue with helper phage (Marks et al. (1992)J. Biol. Chem. 267: 16007-16010) (FIGS. 2A and 2B). Diabody phagemiddisplay a bivalent antibody fragment resulting from intermolecularpairing of one scFv-pIII fusion molecule and one native scFv molecule(FIG. 2B). The C6.5 scFv gene was also subcloned into the phage vectorfd-Sfi/Not. This results in phage with 3 to 5 copies each of scFv-pIIIfusion protein (FIG. 2C). The human breast cancer cell line SKBR3 wasused as a target cell line for endocytosis. Its surface ErbB2 density isapproximately 1.0×10⁶ per cell (Hynes et al. (1989) J. Cell. Biochem 39:167-173).

2) C6.5 Phagemids are Endocytosed by Human Cells

C6.5 scFv phagemids were incubated for 2 hours with SKBR3 cells grown oncoverslips at 37° C. to allow active internalization. Cells wereextensively washed with PBS to remove non specific binding and washed anadditional three times with high salt and low pH (stripping) buffer toremove phage specifically bound to cell surface receptors. Internalizedphagemid were detected with a biotinylated M13 antiserum recognizing themajor coat phage protein pVIII. An anti-botulinum toxin phagemid wasused as a negative control. Staining was analyzed by usingimmunofluorescent microscopy. Approximately 1% of the cells incubatedwith C6.5 scFv phagemid showed a strong intracellular stainingconsistent with endosomal localization while no staining was observedfor anti-botulinum phagemid. Furthermore, no staining was seen if theincubation was performed for 2 hours at 4° C. instead of 37° C. (datanot shown). Staining performed after the PBS washes but before washingwith stripping buffer showed membrane staining of all the cells,indicating that multiple washes with stripping buffer is necessary toremove surface bound phagemids. The results also indicate that only afraction of the cell bound phage are endocytosed.

3) Increased Affinity and Bivalency Lead to Increased Phase Endocytosis

We compared the internalization of C6.5 scFv, C6ML3-9 scFv and C6.5diabody phagemid and C6.5 scFv phage using immunofluorescence. BothC6ML3-9 scFv and C6.5 diabody phagemid as well as C6.5 scFv phageyielded increased intensity of immunofluorescence observed at the cellsurface compared to C6.5 scFv phagemid. For C6ML3-9 scFv phagemid,approximately 10% of the cells showed intracellular fluorescence after 2hours of incubation. This value increased to approximately 30% of cellsfor the dimeric C6.5 diabody phagemid and 100% of cells for multivalentC6.5 scFv phage.

3) Infectious Phase can be Recovered from within the Cell and theirTitre Correlates with the Level of Uptake Observed UsingImmunofluorescence

To determine if infectious phage antibody particles could be recoveredfrom within the cell, we incubated approximately 5.0×10⁵ SKBR-3 cellsfor 2 hours at 37° C. with 3.0×10¹¹ cfu of the different phagemid orphage. Six PBS washes were used to remove non-specifically bound phageand specifically bound phage were removed from the cell surface by threeconsecutive washes with stripping buffer (washes I, II and IIIrespectively, Table 6). The cells were then lysed with 1 mL of a 100 mMtriethylamine solution (TEA) (representing the intracellular phage). Thethree stripping washes and the cell lysate were neutralized and theirphage titer was determined by infection of E. coli TG1. The titers ofphage recovery are reported in Table 6.

TABLE 6 Titration of membrane bound and intracellular phage.Intracellular Cell Surface Phage Titer (×10⁻⁵) Phage Titer PhageAntibody 1st Wash 2nd Wash 3rd Wash (×10⁻⁵) Anti-botulinum 80 6 .8 15phagemid C6.5 scFv 00 6 .6 52 phagemid C6ML3-9 scFv 500 40 2 270phagemid C6.5 diabody 800 20 3 450 phagemid C6.5 scFv phage 300 20 62200 3.0 × 10¹¹ cfu of monovalent C6.5 scFv phagemid, 16 fold higheraffinity monovalent C6ML3-9 scFv phagemid, bivalent C6.5 diabodyphagemid or multivalent C6.5 fd phage were incubated with sub confluentSKBR3 cells for 2 hours at 37° C. Cells were washed 6 times with PBS, 3times with stripping buffer and then lysed to recover intracellularphage. The various fractions were neutralized and the phage titered. Thetotal number of cfu of each fraction is reported. Non specificanti-botulinum phagemid were used to determine non specific recovery.

Considerable background binding was observed in the first stripping washfor the anti-botulinum phage even after 6 PBS washes (2.8×10⁷ cfu, Table6). This value likely represents phage non-specifically bound to thecell surface as well as phage trapped in the extracellular matrix. Theamount of surface bound phage increased only 2.1 fold above thisbackground for C6.5 scFv phagemid (Tables 6 and 7). With increasingaffinity and avidity of the displayed C6.5 antibody fragment, the titerof cell surface bound phagemid or phage increased (Table 6). The titerof phage in the consecutive stripping washes decreased approximately 10fold with each wash. These additional stripping washes led to a minorincrease in the titer of specific phage eluted compared to thebackground binding of the anti-botulinum phage (2.7 fold for C6.5 scFvphagemid to 20 fold for C6.5 scFv phage, Table 7). The only exceptionwas the titer of the C6.5 diabody phagemid, where the ratio actuallydecreased from 6.4 fold to 4.6 fold. This is likely due to the fact thatin the diabody the V_(H) and V_(L) domains that comprise a singlebinding site are not covalently attached to each other via the peptidelinker. This increases the likelihood that a stringent eluent (likeglycine) could dissociate V_(H) from V_(L) and abrogate binding toantigen.

TABLE 7 Specific enrichment of anti-ErbB2 phage compared toanti-botulinum phage. Anti-ErbB2/Anti-Botulinum Phage Titer Ratio*Intracellular/ Cell surface Cell surface Intra- Cell Surface PhageAntibody (1st Wash) (3rd Wash) cellular Phage Ratio** C6.5 scFv 2.14 2.73.5 6.8 phagemid C6ML3-9 scFv 8.9 11.4 18 8.4 phagemid C6.5 diabody 6.44.6 30 35 phagemid C6.5 scFv phage 8.2 20 146 39 *The titers ofanti-ErbB2 phage are divided by the titers of the anti-botulinum phage(Table 6) to derive an enrichment ratio for specific vs nonspecificbinding or internalization. **The titer of intracellular phage isdivided by the titer of cell surface bound phage (Table 6) to derive theratio of internalized phage vs surface bound phage.

Three stripping washes were required to ensure that the titer of phagerecovered after cell lysis was greater than the titer in the laststripping wash (Table 6). We presumed that after three stripping washes,the majority of the phage eluted represented infectious particles fromwithin the cell rather than from the cell surface. In fact, since thecell lysate titer observed with non-specific anti-botulinum phage wasconsiderable (1.5×10⁶) and greater than observed in the last strippingwash, it is likely that many phage remain trapped within theextracellular matrix and relatively inaccessible to the stripping bufferwashes. Some anti-botulinum phage might also be non-specificallyendocytosed by cells, but this is likely to be a small amount given theimmunofluorescence results. The titer of phage in the TEA fractionincreased with increasing affinity and avidity of C6.5, with the highesttiters observed for the dimeric C6.5 diabody phagemid and themultivalent C6.5 scFv phage (Table 6). The values represent a 30 fold(C6.5 diabody phagemid) and 146 fold (C6.5 scFv phage) increase in titercompared to the anti-botulinum phage (Table 6). We have presumed thatthe increase in the phage titer in the cell lysate compared to the laststripping wash is due to endocytosed phage. In fact, some of these phagecould have come from the cell surface or intracellular matrix. Whilethis could be true for a fraction of the phage from the cell lysate, theimmunofluorescence results indicate that at least some of the phage areendocytosed. One indicator of the relative fraction of endocytosed phagefor the different C6.5 molecules is to compare the amount of phageremaining on the cell surface prior to cell lysis (last stripping wash)with the amount recovered after cell lysis. This ratio shows only aminor increase for monovalent C6.5 scFv or C6ML3-9 scFv phagemid (6.8and 8.4 fold respectively) compared to anti-botulinum phagemid (5.4)(Table 7). In contrast the ratios for dimeric C6.5 diabody phagemid andmultivalent C6.5 scFv phage increase to a greater extent (35 and 39respectively) compared to anti-botulinum phagemid.

4) Increasing the Enrichment Ratios of Specifically Endocytosed Phase

The results above indicate that phage antibodies can undergo receptormediated endocytosis and remain infectious in a cell lysate. Selectionof internalized phages from a phage library requires the optimization ofthe method to increase enrichment of specifically internalized phagesover non-internalized phage. Two parameters can be improved: (1)reduction of the recovery of non-specific or non-internalized phage and(2) preservation of the infectivity of internalized phage. To examinethese parameters, we studied wild-type C6.5 scFv phagemid. We chose thismolecule because it was clearly endocytosed based on confocalmicroscopy, yet was the molecule undergoing the least degree of specificendocytosis. C6.5 scFv phagemid also represents the most commonlyutilized format for display of non-immune phage antibody libraries(single copy pIII in a phagemid vector) and has an affinity (16 nM) moretypical of Kd's of scFv from such libraries than the affinity maturedC6ML3-9 scFv (Sheets et al. (1998) Proc. Natl. Acad. Sci. USA 95:6157-6162; Vaughan et al. (1996) Nature Biotech. 14: 309-314).

a) Reducing the Background of Non-Internalized Phase

To reduce the background of non-specific phage recovery, we studied theeffect of trypsinizing the cells prior to TEA lysis. This should removephage trapped in the extracellular matrix. Trypsinization alsodissociates the cells from the cell culture flask, permitting transferto a new vessel and elimination of any phage bound to the cell cultureflask. For these experiments, C6.5 scFv phagemid (5.0×10⁸ ampicillinresistant cfu) were mixed with a 1000 fold excess of wild type fd phage(5.0×10¹¹ tetracylcine resistant cfu). After incubation of phagemid withSKBR-3 cells for 2 hours at 37° C., cells were washed with PBS and threetimes with stripping buffer. Cells were then directly lysed with TEA ortreated with trypsin, washed twice with PBS and then lysed with TEA.Phagemid in the first stripping wash and the cell lysate were titered byinfection of E. coli TG1 and plated on ampicillin and tetracyclineplates. The titer of fd phage and C6.5 scFv phagemid recovered from thecell surface was comparable for the two experimental groups (FIG. 3).The ratio of fd phage/C6.5 scFv phagemid in the cell surface fractions(160/1 and 250/1) yields a 4 to 6 fold enrichment achieved by specificcell surface binding from the initial 1000 fold ratio. Withouttrypsinization, the ratio of fd phage/C6.5 scFv phagemid in the celllysate increases only 6.1 fold; in contrast, the ratio increases 209fold with trypsinization (FIG. 3). This results from a 60 fold reductionin non-specific binding with only a minor reduction in the amount ofspecific phage recovery (FIG. 3).

b) Improving the Recovery of Infectious Internalized Phage

To increase the recovery of infectious internalized phage, we studiedwhether prevention of lysosomal acidification through the use ofchloroquine would protect endocytosed phages from endosomal degradation(Barry et al. (1996) Nat. Med. 2: 299-305). SKBR3 cells were incubatedwith chloroquine and either C6.5 scFv phagemid or anti-botulinumphagemid. Cell lysates were titered at various time points to determinethe number of intracellular phagemid. C6.5 scFv phagemid were present atthe 20 minute time point and the amount of phagemid was comparable withor without the addition of chloroquine. At later time points,approximately twice as much infectious phagemid was recovered with theuse of chloroquine. In contrast, much lower amounts of anti-botulinumphage were present and chloroquine had no effect on the titer,suggesting that the phagemid result from non-specific surface bindingrather than non-specific endocytosis into endosomes. Overall, theresults indicate that prevention of lysosomal acidification increasesthe amount of infectious phage recovered for incubations longer than 20minutes.

5) Recovery of Internalized Phase at Low Phase Concentrations

Only very large phage antibody libraries containing more than 5.0×10⁹members are capable of generating panels of high affinity antibodies toall antigens (10, 23, 24). Since phage can only be concentrated toapproximately 10¹³ cfu/ml, a typical phage preparation from a largelibrary will only contain 104 copies of each member. Thus selection oflibraries for endocytosis could only work if phage can be recovered whenapplied to cells at titers as low as 10⁴. We therefore determined therecovery of infectious phage from within SKBR3 cells as a function ofthe phage titer applied. SKBR3 cells were incubated with C6.5 scFv,C6ML3-9 scFv or C6.5 diabody phagemids or C6.5 scFv phage for 2 hours at37° C. Cells were washed three times with stripping buffer, trypsinizedand washed twice with PBS. Cells were lysed and intracellular phagetitered on E. coli TG1. Phage recovery increased with increasing phagetiter for all phage studied (FIG. 5). For monovalently displayedantibodies, phagemid could not be recovered from within the cell atinput titers less than 3.0×10⁵ (C6.5 scFv) to 3.0×10⁶ (C6ML3-9 scFv)This threshold decreased for bivalent and multivalent display (3.0×10⁴for C6.5 diabody phagemid and C6.5 scFv phage).

C) Discussion

We demonstrate for the first time that phage displaying an anti-receptorantibody can be specifically endocytosed by receptor expressing cellsand can be recovered from the cytosol in infectious form. The resultsdemonstrate the feasibility of directly selecting internalizingantibodies from large non-immune phage libraries and identify thefactors that will lead to successful selections. Phage displayinganti-ErbB2 antibody fragments are specifically endocytosed by ErbB2expressing SKBR3 cells, can be visualized within the cytosol and can berecovered in an infectious form from within the cell. When monovalentscFv antibody fragments were displayed monovalently in a phagemidsystem, recovery of internalized phage was only 3.5 to 18 fold abovebackground. Display of bivalent diabody or multivalent display of scFvin a phage vector increased recovery of internalized phage to 30 to 146fold above background. This result is consistent with our studies ofnative monomeric C6.5 scFv and dimeric C6.5 diabody as well as studiesof other monoclonal anti-ErbB2 antibodies where dimeric IgG but notmonomeric Fab dimerize and activate the receptor and undergo endocytosis(Yarden (1990) Proc. Natl. Acad. Sci. USA 87: 2569-2573; Hurwitz et al.(1995) Proc. Natl. Acad. Sci. USA 92: 3353-3357). In fact it is likelythat endocytosis of C6.5 and C6ML3-9 scFv phagemids reflect the smallpercentage of phage displaying two or more scFv (Marks et al. (1992) J.Biol. Chem. 267: 16007-16010). The importance of valency in mediatingeither high avidity binding or receptor crosslinking and subsequentendocytosis is confirmed by the only other report demonstrating specificphage endocytosis. Phage displaying approximately 300 copies of a highaffinity Arg-Gly-Asp integrin binding peptide on pVIII were efficientlyendocytosed by mammalian cells (Hart et al. (1994) J. Biol. Chem. 269:12468-12474). Recovery of phage after endocytosis also increases thespecificity of cell selections compared to recovery of phage from thecell surface. Thus enrichment ratios for specific vs non-specificsurface binding range from 2 to 20 fold. These values are comparable tothe approximately 10 fold enrichment reported by others for a singleround of cell surface selection (Pereira et al. (1997) J. Immunol. Meth.203: 11-24; Watters et al. (1997) Immunotechnology 3: 21-29). Incontrast our enrichment ratios for specific vs non-specific endocytosisrange from 3.5 to 146 fold.

Based on these results, selection of internalizing antibodies from phageantibody libraries would be most successful with either homodimericdiabodies in a phagemid vector or multivalent scFv using a phage vector.While no such libraries have been published, there are no technicalbarriers preventing their construction. Multivalent libraries wouldpresent the antibody fragment in the form most likely to crosslinkreceptor and undergo endocytosis. Antibodies from such libraries wouldneed to be bivalent to mediate endocytosis. Alternatively, monomericreceptor ligands can activate receptors and undergo endocytosis, eitherby causing a conformational change in the receptor favoring the dimericform or by simultaneously binding two receptors. Monomeric scFv thatbound receptor in a similar manner could also be endocytosed. Thusselection of libraries of monovalent scFv in a phagemid vector couldresult in the selection of ligand mimetics that activate receptors andare endocytosed as monomers. Such scFv could be especially useful forthe construction of fusion molecules for the delivery of drugs, toxinsor DNA into the cytoplasm. Since antibodies which mediate receptorinternalization can cause receptor down regulation and growth inhibition(Hurwitz et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3353-3357; Hudziaket al. (1989) Mol. Cell. Biol. 9: 1165-1172; Stancovski et al. (1991)Proc. Natl. Acad. Sci. USA 88: 8691-8698; Lewis et al. (1993) CancerImmunol. Immunother. 37: 255-263), selection for endocytosableantibodies may also identify antibodies which directly inhibit ormodulate cell growth.

Example 5 Transfection of Cells

The F5 scFv gene was removed from pHEN1-F5 by digestion of phagemid DNAwith the restriction enzymes SfiI and NotI. A phage vector based onFdDOG1 (See prior Ref.), but modified to insert an SfiI site into thegene III leader sequence, was digested with SfiI and NotI and thedigested F5 gene ligated into digested phage Fd vector DNA. Recombinanttransformant were identified. E. coli containing the F5 recombinantphage were grown in culture to produce F5-Fd phage (see Maniatis forphage preparation). F5 phages were then used to infect E. coli harboringa phagemid which contains a mammalian promoter (CMV) followed by eitherthe gene for ∃-galactosidase (pcDNA3.1/HisB/LacZ, In Vitrogen) or thegene for the enhanced green fluorescent protein (pN2EGFP, Clonetchplasmid) and a eucaryotic polyadenylation sequence. Bacteria were grownovernight in the presence of tetracycline 15 ug/mL and either ampicillin100 ug/mL (pcDNA3.1/HisB/LacZ containing bacteria) or Kanamycine 30ug/mL (pN2EGFP containing bacteria). The phage prepared from thesupernatant a mixture of F5-Fd coat contains either the reporter gene(about 50% of the phages) in a single strand format or the F5-Fd phagegenome (about 50% of the phages). Incubation of ErbB2 positive cells5.105 SKBR3 with 10⁷ pfu the phage mix (Filtered twice through a 0.45 nmfilter to sterility) allowed expression of the reporter gene in 1% ofthe cells. Cells incubated with an 10 time fold more negative controlphage, i.e. reporter gene packaging in wild type Fd, showed noexpression of the reporter genes. In an experiment where a mixedpopulation of ErbB2 high (SKBR3) and ErbB2 low cells (MCF7) (Lewis etal. (1993) Cancer Immunol Immunother 37: 255-263) were incubated withthe F5-Fd-EGFP phages for two days, we obtained the expression of thereporter gene only in erbB2 positive cells, cells being differentiatedby their ErbB2 level by FACS.

Example 6 Targeted Gene Delivery to Mammalian Cells by FilamentousBacteriophage

In this example we show that prokaryotic viruses can be re-engineered toinfect eukaryotic cells resulting in expression of a portion of thebacteriophage genome. Phage capable of binding mammalian cellsexpressing the growth factor receptor ErbB2 and undergoing receptormediated endocytosis were isolated by selection of a phage antibodylibrary on breast tumor cells and recovery of infectious phage fromwithin the cell. As determined by Immunofluorescence, F5 phage wereefficiently endocytosed into 100% of ErbB2 expressing SKBR3 cells. Toachieve expression of a portion of the phage genome, F5 phage wereengineered to package the green fluorescent protein (GFP) reporter genedriven by the CMV promoter. These phage when applied to cells underwentErbB2 mediated endocytosis leading to GFP expression. GFP expressionoccurred only in cells overexpressing ErbB2, was dose dependent reaching4% of cells after 60 hours and was detected with phage titers as low as2.0×10⁷ cfu/ml (500 phage/cell). The results demonstrate that bacterialviruses displaying the appropriate antibody can bind to mammalianreceptors and utilize the endocytic pathway to infect eukarotic cellsresulting in viral gene expression. This represents a novel method todiscover targeting molecules capable of delivering a geneintracellularly into the correct trafficking pathway for gene expressionby directly screening phage antibodies. This should significantlyfacilitate the identification of appropriate targets and targetingmolecules for gene therapy or other applications where delivery into thecytosol is required. This approach can also be adapted to directlyselect, rather than screen, phage antibodies for targeted geneexpression. The results also demonstrate the potential of phageantibodies as an in vitro or in vivo targeted gene delivery vehicle.

A) Introduction

Widespread application of gene therapy requires the ability to target atherapeutic gene to the appropriate cell or tissue type with highefficiency (Michael and Curiel (1994) Gene Ther. 1: 223-232). Targetingof retroviral vectors has been reported by inserting receptor ligands orsingle chain Fv (scFv) antibody fragments into the viral envelopeprotein (Kasahara et al. (1994) Science 266: 1373-1376). Targeting ofadenoviral vectors has been achieved by use of ‘adapter’ fusionmolecules consisting of an antibody fragment which binds the adenoviralknob and a cell targeting molecule such as a receptor ligand or antibody(Douglas et al. (1996) Nat. Biotechnol. 14: 1574-1578; Watkins et al.1997) Gene Ther. 4(10): 1004-1012). Targeting of non-viral vectors usingcell surface receptor ligands or antibodies has also been reported(Fominaya and Wels (1996) J. Biol. Chem. 271(18): 10560-10568; Michaeland Curiel (1994) Gene Ther. 1: 223-232). All of these approaches dependon the use of targeting molecules which bind a cell surface receptorresulting in internalization of the gene delivery vehicle withsubsequent delivery of the DNA to the nucleus. Identification ofappropriate targeting molecules has largely been performed byindividually screening receptor ligands or antibodies. In the case ofscFv antibody fragments this typically requires construction of the scFvfrom the V-genes of a hybridoma, construction of the targeted genedelivery vehicle, and in vitro evaluation of targeting ability.

More recently, it has proven possible to directly select peptides andantibody fragments binding cell surface receptors from filamentous phagelibraries (Andersen et al. (1996) Proc. Natl. Acad. Sci. USA 93(5):1820-1824; Barry et al. (1996) Nat. Med. 2: 299-305; Cai and Garen(1995) Proc. Natl. Acad. Sci. USA 92(24): 6537-6541; de Kruif et al.(1995) Proc. Natl. Acad. Sci. USA 92(6): 3938-3942; Marks et al. (1993)Bio/Technology 11(10): 1145-1149). This has led to a marked increase inthe number of potential targeting molecules. The ability ofbacteriophage to undergo receptor mediated endocytosis (Barry et al.(1996) Nat. Med. 2: 299-305; Hart et al. (1994) J. Biol. Chem. 269(17):12468-12474) indicates that phage libraries can be selected not only forcell binding but also for internalization into mammalian cells. If thephage single stranded phage genome can be transcribed and translated,then it should prove possible to screen or select for phage which bindreceptors in a manner which leads to endocytosis and delivery of thephage genome into the correct trafficking pathway leading to expression.It has been previously shown that phage can enter mammalian cells afterchemical alteration of the cell membrane leading to reporter geneexpression (Okayama and Berg (1985) Mol. Cell. Biol. 5(5): 1136-1142;Yokoyama-Kobayashi and Kato (1993) Biochem. Biophys. Res. Commun.193(2): 935-939). More recently, Larocca et al. showed that indirectbacteriophage mediated gene delivery could occur by targetingbiotinylated phage via streptavidin and biotinylated fibroblast growthfactor (FGF) to mammalian cells expressing FGF receptor (Larocca et al.(1998) Hum. Gene Ther. 9: 2393-2399).

In this report, we show that filamentous phage displaying the anti-ErbB2scFv F5 as a genetic fusion with the phage minor coat protein pill candirectly infect mammalian cells expressing ErbB2 leading to expressionof a reporter gene contained in the phage genome. This offers a new wayto discover targeting molecules for intracellular drug delivery or genetherapy by directly screening phage antibodies to identify those capableof undergoing endocytosis and delivering a gene intracellularly into thecorrect trafficking pathway for gene expression. This shouldsignificantly facilitate the identification of appropriate targets andtargeting molecules for gene therapy or other applications wheredelivery into the cytosol is required. We also discuss how this approachmight be used to directly select phage antibodies for targeted geneexpression. Finally, we discuss the potential for use of phageantibodies themselves for in vitro or in vivo targeted gene deliveryvectors.

B) Materials and Methods

1) Anti-ErbB2 F5 scFv

An anti-ErbB2 scFv (F5) in the vector pHEN-1 (Hoogenboom et al. (1991)Nucleic Acids Res. 19(15): 4133-4137) (pHEN-F5) was obtained byselecting a non-immune phage antibody library (Sheets et al. (1998)Proc. Natl. Acad. Sci. USA 95(11): 6157-6162) on ErbB2 expressing SKBR3cells followed by screening for binding on recombinant ErbB2extracellular domain (ECD). The native F5 scFv binds ErbB2 ECD with aK_(d)=1.6×10⁻⁷ M as determined by surface plasmon resonance in a BIAcoreas previously described (Schier et al. (1996) J. Mol. Biol. 255(1):28-43).

2) Phage and Phagemid Vectors

pcDNA3-GFP (6.1 Kbp) was obtained by subcloning the Hind III/Not Ifragment of pN2EGFP (4.7 Kbp) (Clontech) into the pcDNA3-HisB/LacZ(Invitrogen) Hind III/Not I backbone. A fd-F5-phage vector wasconstructed by subcloning the Sfi I/Not I scFv-F5 insert from pHEN-1into the Sfi I/Not I sites of fd-Sfi/Not (constructed from fd-tet-DOG(Clackson et al. (1991) Nature 352(6336): 624-628) by changing the ApaL1cloning site in the gene III leader to SfiI. The pHEN-F5-GFP phagemidvector (6.8 Kbp) was obtained by subcloning the 1.6 Kbp pN2EGFP bluntedAse I/Afl II fragment into the blunted EcoR I site of pHEN-F5. Theorientation of the insert was analyzed by Not I restriction digest.

3) Cell Line Culture and Transfection

SKBR3 and MCF7 were grown in RPMI complemented with 10% fetal bovineserum (FBS) (Hyclone). 50% confluent SKBR3 cells grown in 6-well plateswere transfected with 1 μg of DNA per well using Lipofectamine™ (GIBCOBRL) as recommended by the manufacturer. pN₂EGFP dsDNA was prepared byalkaline lysis using the Maxiprep Qiagen Kit (Qiagen Inc.). ssDNA wasextracted from 500 μl of phagemid preparation (see below) by 2 phenolextractions followed by ethanol precipitation. DNA was quantified byspectophotometry with 1.0 A₂₆₀ nm equal to 40 μg/ml for ssDNA or 50μg/ml for dsDNA. For GFP detection, cells were detached using atrypsin-EDTA mix (GIBCO BRL) and analyzed on a FACScan™ (BectonDickinson).

4) Phagemid and Phase Preparation

pHEN-F5, pHEN-F5-GFP, pcDNA3-GFP or pN2EGFP phagemids were prepared fromE. coli TG1 by superinfection with VCS-M13 helper phage (Stratagene) aspreviously described (Marks et al. (1991) J. Mol. Biol. 222(3):581-597). Fd-F5-phage were prepared from E. coli TG1 as previouslydescribed (McCafferty et al. (1990) Nature 348(6301): 552-554).F5-GFP-phage and F5-LacZ-phage were prepared by superinfection of E coliTG1 containing pcDNA3-GFP with fd-F5-phage. Virus particles werepurified from the culture supernatant by 2 polyethylene glycolprecipitations (Sambrook et al. (1990). Molecular cloning—a laboratorymanual, Cold Spring Harbor Laboratory, New York) resuspended inphosphate buffered saline, pH 7.4 (PBS), filtered through a 0.45 μmfilter and stored at 4° C. Alternatively, the preparations weresubmitted to an additional CsCl ultracentrifugation step (Smith andScott (1993) Meth. Enzymol. 217:228-257). The ratio of packaged helperphage DNA versus phagemid DNA was determined by titering (Sambrook etal., supra.) the phage for ampicillin and kanamycin resistance (forhelper phage rescued pHEN-F5) or ampicillin and tetracycline resistance(for fd-F5 phage rescued pcDNA3-GFP).

5) Phage FACS

Cells were trypsinized, washed with PBS containing 1% FBS (FACS buffer)and resuspended at 10 cells/ml in the same buffer. The stainingprocedure was performed on ice with reagents diluted in FACS buffer. Onehundred μl aliquots of cells were distributed in conical-96-well plate(Nunc), centrifuged at 300g and the cell pellets resuspended in 100 μlof serial dilutions of phage or phagemid preparation and incubated for 1hr. Cells were centrifuged and washed twice, the cell pelletsresuspended in 100 μl of anti-M13 antibody (5 Prime, 3 Prime Inc.)(diluted 1/400) and incubated for 45 min. Cells were washed as above,resuspended in 100 μl of streptavidin-Phycoerythrin (Jackson Inc.)(diluted 1/400) and incubated for 20 min. After a final wash, the cellswere analyzed by FACS.

6) Immunofluorescence

Cells were grown on coverslips to 50% confluency in 6 well-plates. Phagepreparation (less than 10% of the culture medium) was added and thecells were incubated for 16 hours. The coverslips were washed 6 timeswith PBS, 3 times for 10 min with Glycine buffer (50 mM glycine, pH 2.8,NaCl 500 mM), neutralized with PBS and fixed with PBS-4%paraformaldehyde for 5 min at room temperature. Cells were permeabilizedwith cold acetone for 30 sec, saturated with PBS-1% BSA and incubatedwith anti-M13 antibody (d: 1/300 in the saturation solution) followed bystreptavidin-Texas Red (Amersham) (d: 1/300 in the saturation solution).Coverslips were analyzed with an Axioskop fluorescent microscope(Zeiss).

7) Bacteriophage Mediated Cell Infection

CsCl phage preparations were diluted at least 10 fold in cell culturemedium, filtered through a 0.45 μm filter and added to 30% to 80%confluent cells. After incubation, the cells were trypsinized, washedwith FACS buffer and analyzed for GFP expression by FACS. In theexperiments where MCF7 and SKBR3 were co-cultured, ErbB2 expression wasquantitated by FACS using the anti-ErbB2 mouse mAb 4D5 which binds ErbB2ECD (10 μg/ml) (1 hr), biotinylated sheep anti-mouse immunoglobulins(Amersham) and streptavidin-Phycoerythrin.

C) Results

1) Internalization of ErbB2 Binding Monovalent and Multivalent F5 PhaseParticles by ErbB2 Expressing Cells

We isolated the anti-ErbB2 scFv-F5 from a library of scFv displayed onthe surface of bacteriophage as fusions to pIII (Sheets et al. (1998)Proc. Natl. Acad. Sci. USA 95(11): 6157-6162) by selection on ErbB2expressing SKBR3 breast tumor cells and recovery of infectious phagefrom within the cell (M. Poul et al., manuscript in preparation). Thisselection strategy was employed to select scFv capable of undergoingendocytosis upon receptor binding. When the pHEN-F5 phagemid vector isrescued with VCS-M13 helper phage, the resulting virus particles(F5-phagemid) display an average of 1 copy of scFv-pIII fusion proteinand 3 to 4 copies of the wild type pIII minor coat protein from thehelper phage (Marks et al. (1992) J. Biol. Chem. 267(23): 16007-16010).As a result, the phagemid bind monovalently. To improve the binding ofthe virus particles to ErbB2 expressing cells, multivalent phageantibodies were created by subcloning the F5 scFv DNA into the phagevector fd-Sfi/Not for fusion with the pIII protein. Virus particles,referred to as fd-F5 phage, display 4 to 5 copies of scFv-pIII fusionprotein (Id.).

To determine whether F5 phage antibodies could be internalized bymammalian cells, SKBR3 cells overexpressing ErbB2 were incubated for 16hrs with fd-F5 phage (10⁹ colony forming unit/ml, cfu/ml), F5 phagemid(10¹¹ cfu/ml), or with phagemids displaying an irrelevant anti-botulinumscFv-pIII fusion protein (10¹² cfu/ml) (Amersdorfer et al., 1997) as anegative control. The cell surface was stripped of phage antibodiesusing low pH glycine buffer, the cells permeabilized and fixed, andintracellular phage detected with anti-M13 antibody. Remarkably, allcells showed strong intracellular staining when incubated with fd-F5phage or with F5 phagemid but not when incubated with the anti-botulinumphagemid. This demonstrates the dependence of phage entry on thespecificity of the scFv fused to pIII.

2) Preparation of ErbB2 Binding Phases and Phagemids Packaging aReporter Gene for Expression in Eukaryotic Cells

Two strategies were used to investigate whether F5 phage could deliver areporter gene to mammalian cells leading to expression. To makemonovalent phage containing a reporter gene, we cloned the gene forgreen fluorescent protein (GFP) driven by the CMV promoter into thephagemid vector pHEN-F5 generating the vector pHEN-F5-GFP (FIG. 6, leftpanel). Escherichia. coli TG1 containing pHEN-F5-GFP (ampicillinresistant) were infected with helper phage (kanamycin resistant) andhigh titers of monovalent F5-GFP phagemids were obtained (5.0×10¹⁰ampicillin resistant cfu/ml of culture supernatant). The ratio ofpackaged phagemid DNA versus helper phage DNA (ampicillin versuskanamycin resistant cfu) was determined to be 100:1. To make multivalentphage containing a reporter gene, fd-F5-GFP phage were generated byinfecting E. coli TG1 carrying the pcDNA3-GFP phagemid (ampicillinresistant) with fd-F5 phage (tetracycline resistant), thus using fd-F5phage as a helper phage. The fd-F5-GFP phage titer was approximately5.0×10⁸ ampicillin resistant cfu/ml of culture supernatant. Lower phagetiters result when fd is used as a helper phage because it lacks aplasmid origin of replication leading to interference from the phagemidf1 origin (Cleary and Ray (1980) Proc. Natl. Acad. Sci. USA77(8):4638-4642). The ratio of packaged reporter gene DNA versus phageDNA (ampicillin versus tetracycline resistant cfu) was 1:1. The lowerratio of reporter gene/helper genome when using fd as a helper phage isdue to the presence of a fully functional packaging signal on the fdgenome compared to the mutated packaging signal in VCS-M13 (Vieira andMessing (1987) Meth. Enzymol. 153: 3-11). Both phage and phagemidpreparations were assessed for SKBR3 cell binding (FIG. 7). While bothpreparations bound SKBR3 cells, binding could be detected with as littleas 10⁸ cfu/ml of fd-F5-GFP phage cfu/ml (160 femtomolar) compared to10¹⁰ cfu/ml of F5-GFP phagemids (15 picomolar). Thus multivalent bindingleads to an increase in the apparent binding constant of virusparticles.

3) Targeted Phagemid and Phase Mediated Gene Transfer into ErbB2Expressing Breast Cancer Cells

To determine if ErbB2 binding phagemids were capable of targeted genedelivery, 2.0×10⁵ SKBR3 cells (a breast tumor cell line expressing highlevels of ErbB2) or 2.0×10⁵ MCF7 cells (a low ErbB2 expressing breasttumor cell line) were incubated with 5.0×10¹¹ cfu/ml F5-GFP phagemids at37° C. Cells were analyzed for GFP expression by FACS after 48 hrs (FIG.8A). 1.37% of the SKBR3 cells expressed GFP after incubation with F5-GFPphagemids (FIG. 8A6). GFP expression was identical regardless of theorientation of the f1 packaging signal (data not shown), indicating thattranscription/translation was proceeding via synthesis of thecomplementary DNA strand. GFP expression was not detected in SKBR3 cellsincubated with no phage or with helper phage packaging the reporter gene(FIG. 8A4 and 8A5). Expression was also not seen in MCF7 cells incubatedwith no phage, helper phage or pHEN-F5-GFP, indicating the requirementof ErbB2 expression for targeted gene delivery (FIG. 8A1, 8A2 and 4A3).Since gene transfer applications are likely to involve targeting ofspecific cells in an heterogeneous cell population, we performed thesame experiment on a co-culture of SKBR3 and MCF7 cells (FIG. 8B). Cellswere stained for ErbB2 expression to discriminate MCF7 from SKBR3 cellsand the GFP expression of each subpopulation was analyzed by FACS. OnlySKBR3 cells (1.91%) expressed GFP. Similar results were found usingF5-GFP phages instead of F5-GFP phagemids (data not shown). These dataconfirm that fd-F5-GFP phage and F5-GFP phagemid mediated gene deliveryis restricted to ErbB2 overexpressing cells and can be targeted to suchcells in the presence of non-expressing cells.

4) Characterization of Phage Mediated Gene Transfer

To determine the dose-response characteristics of phage mediated genetransfer, SKBR3 cells were incubated for 60 hrs with increasing amountsof fd-F5-GFP phage or F5-GFP phagemids and the percent of GFP positivecells determined (FIGS. 9A and 9B). The minimal phage concentrationrequired for detection of a significant number of GFP positive cells(FIG. 9A) was approximately 4.0×10⁷ cfu/ml for fd-F5-GFP phage (0.13%)and 1.0×10¹⁰ cfu/ml for F5-GFP phagemid (0.12%). The values correlateclosely with the binding curves (FIG. 7) and indicate that multivalentphage are 100 to 1000 time more efficient than phagemids in terms ofgene expression. No significant number of positive cells were observedwith up to 4.0×10¹³ cfu/ml of helper phage packaging the reporter gene.For both phage and phagemid, the percent of GFP positive cells increasedwith phage concentration with no evidence of a plateau. The maximumvalues achieved were 2% of cells for fd-F5-GFP phage and 4% for F5-GFPphagemids and appear to be limited by the phage titer applied (1.5×10⁹cfu/ml and 4.0×10¹² cfu/ml respectively). The amount of GFP expressedper cell (estimated by the mean fluorescent intensity (MFI), FIG. 9B)also increased with phage concentration, with a small number of cellsshowing expression with phage titers as low as 2.0×10⁷ cfu/ml (fd-F5-GFPphage) to 1.0×10¹⁰ cfu/ml (F5-GFP phagemid).

To compare the yield of gene expression obtained with phage totraditional transfection methods, single stranded (ssDNA) or doublestranded (dsDNA) was transfected into SKBR3 using lipofectamine. Per μgof ss DNA, efficiency of phagemid mediated gene delivery (approximately1%) was comparable to lipofectamine transfection of ssDNA (0.98%) anddsDNA (1.27%) (Table 8). Efficiency was approximately 500 fold higherfor phage mediated transfection, with 2.25 ng of ss DNA resulting intransfection of 0.87% of cells.

TABLE 8 Transfection efficiencies in SKBR3 cells. % of GFP TransfectionAmount of reporter positive method Reporter plasmid plasmid DNA cells*F5-phagemid pHEN-F5-GFP 15 μg 3.84 Mediated 3.1 μg 1.44 0.78 μg 0.64fd-F5-phage pcDNA3-GFP 5 ng 1.69 mediated 2.25 ng 0.87 1.25 ng 0.57Helper phage pN₂GFP 100 μg 0.12 mediated 20 μg 0.07 5 μg 0.06Lipofectamine pN₂GFP dsDNA 1 μg 1.27 ssDNA 1 μg 0.98 *Cells wereanalysed 48 hours after transfection for GFP expression using FACS.Results are expressed in % of GFP positive cells. **For phage, theamount of reporter plasmid was calculated from the plasmid size and thenumber of ampicillin (pHEN-F5-GFP or pcDNA3-GFP) or kanamycin (pN₂GFP)resistant colonies. Mock transfected cells contained an average of 0.05%GFP positive cells.

To determine the time course of gene expression, 5.0×10¹¹ cfu/ml ofF5-GFP phagemid were incubated with SKBR3 cells. After 48 hrs, theculture medium was replaced by fresh medium. GFP expressing cells can bedetected within 24 hrs after phage are applied and the percentage ofpositive cells increases linearly with increasing time to a maximum of4.5% by 120 hours (FIG. 9C). The GFP content of the positive cells, asestimated by the MFI, increases up to 96 hrs (FIG. 9D). After 96 hrs,the number of GFP positive cells continues to increase but the MFIdecreases, probably due to the repartition of GFP molecules to daughtercells during cell division.

C) Discussion

We demonstrate that filamentous phage displaying an anti-ErbB2 scFvantibody fragment as a genetic fusion with the minor coat protein pIIIcan be directly targeted to mammalian cells expressing the specificityof the scFv. Such phage undergo receptor mediated endocytosis and enteran intracellular trafficking pathway which ultimately leads to reportergene expression. This is a remarkable finding demonstrating thatprokaryotic viruses can be re-engineered to infect eukaryotic cellsresulting in expression of a portion of the bacteriophage genome. Geneexpression was detected with as few as 2.0×10⁷ cfu of phage andincreased with increasing phage titer up to 4% of cells. Multivalentdisplay decreased the threshold for detectable gene expressionapproximately 500 fold compared to monovalent display, most likely dueto an increase in the functional affinity and an increased rate ofreceptor mediated endocytosis from receptor crosslinking. The maximumpercent of cells transfected, however, was higher for monovalent display(phagemid) due to the significantly higher phage titer generated. Thelower titer of multivalent phage is due to interference of the f1 originof replication on the reporter phagemid with the fd phage antibodyorigin of replication (Cleary and Ray (1980) Proc. Natl. Acad. Sci. USA77(8): 4638-4642).

Targeted infection of mammalian cells using phage which bindendocytosable receptors is likely to be a general phenomenon. Forexample, fusing an anti-transferrin receptor scFv to gene III ofpHEN-GFP results in GFP expression in 10% of MCF7 cells, 4% of SKBR3cells, 1% of LNCaP cells and 1% of primary melanoma cells. Similarly,targeted GFP gene delivery to FGF receptor expressing cells usingbiotinylated phage and a streptavidin-FGF fusion molecule was recentlyreported (Larocca et al. (1998) Hum. Gene Ther. 9: 2393-2399). However,direct genetic fusion of the targeting molecule via gene III may be moreefficient than using adapter molecules. Thus while the maximum percentof cells transfected using the FGF-adapter molecule was not reported, weestimate it to be only 0.03% of FGF expressing L6 rat myoblasts based onthe number of cells infected, the time after infection to themeasurement of gene expression and the number of cells expressing GFP.While a greater frequency of expression (0.5%) was seen in COS-1 cells,this results from the presence of large T antigen and SV40 mediated DNAreplication and thus is not generalizable to most cells.

The approach we describe represents a novel method to discover ligandsfor targeted intracellular drug or gene delivery. Phage antibody orpeptide libraries are first selected for endocytosis by mammalian cells(Barry et al. (1996) Nat. Med. 2: 299-305) or for binding to purifiedantigen, cells, tissues or organs. After subcloning the selected scFvgenes into the pHEN-GFP vector, phage produced from individual coloniescan be directly screened for gene expression. This is possible sinceexpression can be detected with as little as 1.0×10¹⁰ cfu of phagemids.This permits not only direct identification of endocytosed scFv but alsothe subset of receptor antibodies which undergo proper trafficking forgene expression. If multivalent display is necessary for efficientendocytosis, the scFv genes can be subcloned into fd-Sfi-Not which isthen used to rescue the reporter phagemid. Use of scFv-fd phage alsoallows the targeting of a large number of different reporter genes invarious expression vectors since many commercially available mammalianvectors contain f1 origins of replication. As such, antibody targetedphage might prove useful transfection reagents, especially for cellsdifficult to transfect by standard techniques.

It may also prove possible to use this approach to directly select,rather than screen, antibodies for targeted gene delivery. For example,mammalian cells are incubated with a phage antibody library containingthe GFP gene, and then sorted based on GFP expression using FACS. Phageantibody DNA would be recovered from the mammalian cytoplasm by celllysis and used to transfect E. coli and prepare more phage for anotherround of selection. If the quantities of recoverable phage DNA areinadequate, inclusion of the neomycin gene in the pHEN-GFP vector wouldpermit selection of GFP expressing mammalian cells using G418 (Laroccaet al. supra).

Finally, this system has promise as a targetable in vitro or in vivogene therapy vehicle. The main limitations are infection efficiency,pharmacokinetics and immunogenicity. With respect to infectionefficiency, values achieved by targeted phage in this report (8.0×10⁴/mlof phage preparation) are not dissimilar to values reported for targetedretrovirus (10³-10⁵/ml of virus) (Kasahara et al. (1994) Science 266:1373-1376; Somia et al. (1995) Proc. Natl. Acad. Sci. USA 92(16):7570-7574) but less than reported for adenovirus targeting strategies(Douglas et al. (1996) Nat. Biotechnol. 14: 1574-1578; Watkins et al.1997) Gene Ther. 4(10): 1004-1012). The factors limiting higherinfection efficiencies, however, are likely to differ between thesystems. Thus while the percentage of cells infected by retrovirus issignificantly higher than observed for bacteriophage, infection islimited by the problems encountered producing large numbers of viruswhich can enter the cell. Since all cells take up the targeted phage,gene expression is limited by one or several post-uptake events (e.g.degradation of phage to release DNA, endosomal escape, nuclear targetingor transcription). More detailed study of the fate of the phage and itsDNA is likely to suggest where the block lies permitting engineering ofphage to increase infection efficiency. For example, endosomal escapecould be enhanced by co-administering replication defective adenovirus(Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88(19): 8850-8854) orincorporating endosomal escape peptides (Wagner et al. (1992) Proc.Natl. Acad. Sci. USA 89(17): 7934-7938) or proteins (Fominaya and Wels(1996) J. Biol. Chem. 271(18): 10560-10568) into the phage major coatprotein pVIII. Alternatively, infection efficiency could be increasedcombinatorially by creating scFv targeted libraries of pVIII mutants andselecting for increased gene expression. With respect topharmacokinetics, though not extensively studied, it is likely that thebiodistribution of phage is limited to the intravascular space. Thiswould not affect in vitro phage gene therapy, but might limit in vivouses to those targeting the vasculature. This still leaves numerousapplications including those where neovascularization plays a role, suchas cancer. With respect to immunogenicity, it is likely that phage willbe immunogenic, thus limiting the number of times that phage could beadministered in vivo. Alternatively, it might prove possible to evolvethe major coat protein pVIII to reduce or eliminate immunogenicity forexample by negatively selecting a pVIII library on immune serum (Jenneet al. (1998) J. Immunol. 161(6): 3161-3168).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of selecting antibody binding moieties that are internalizedinto target cells, said method comprising: i) contacting one or more ofsaid target cells with one or more members of a polyvalent antibodyphage display library; ii) culturing said target cells underinternalizing conditions; and iii) identifying members of saidpolyvalent antibody phage display library internalized into the one ormore of said target cells.
 2. The method of claim 1, wherein saidpolyvalent phage display library comprises: a) a phage display libraryin which each member phase displays, on average, at least one copy of anantibody-phage coat protein fusion product comprising two or morebinding domains, each said domain including a variable heavy chainregion and a variable light chain region of an antibody; or b) a phasedisplay library in which each member phage displays, at average, two ormore copies of an antibody-phage coat protein fusion product comprisinga variable heavy chain region and a variable light chain region of anantibody.
 3. The method of claim 2, wherein said antibody phage displaylibrary displays single chain antibody Fv regions.
 4. The method ofclaim 1, wherein said identifying comprises recovering internalizedphage and optionally repeating steps (i) through (iii) to further selectfor internalizing binding moieties.
 5. The method of claim 4, whereinsaid recovering comprises: (a) lysing said target cells to releaseinternalized phage; and (b) infecting a bacterial host with saidinternalized phage to produce phage for a subsequent round of selection.6. (canceled)
 7. The method of claim 1, further comprising the step ofcontacting members of said polyvalent antibody phase display librarywith cells of a subtractive cell line.
 8. The method of claim 7, whereinsaid cells of a subtractive cell line are present in at least 2-foldexcess over said target cells.
 9. (canceled)
 10. (canceled)
 11. Themethod of claim 1, wherein step (ii) comprises culturing the targetcells at a temperature of about 35° C. to about 37° C. and a pH fromabout 6 to about
 8. 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. Themethod of claim 1, wherein said target cells are selected from the groupconsisting of solid tumor cells, members of a cDNA expression library,cells that overexpress a cytokine receptor, cells that overexpress agrowth factor receptor, metastatic cells, cells of a transformed cellline, cells transformed with a gene or cDNA encoding a specific surfacetarget receptor, and neoplastic cells derived from outside a solidtumor.
 16. The method of claim 7, wherein said cells of a subtractivecell line are selected from the same tissue type as the target cells.17. The method of claim 7, wherein said cells of a subtractive cell lineare selected from the group consisting of fibroblasts, monocytes, stemcells, and lymphocytes. 18-50. (canceled)
 51. The method of claim 4,wherein said recovering comprises removing polyvalent antibody phagedisplay library members that are not internalized by the one or moretarget cells.
 52. The method of claim 51, wherein said removingcomprises washing the target cells with a strong wash.
 53. The method ofclaim 51, wherein said removing comprises removing the polyvalentantibody phage display library members bound to an extracellular matrixof the target cells.
 54. The method of claim 51, wherein said removingcomprises trypsinization of the target cells.
 55. The method of claim 7wherein said cells of a subtractive cell line are present in at least2-fold excess over said target cells.
 56. The method of claim 7, whereinsaid cells of a subtractive cell line comprise live cells.
 57. Themethod of claim 7, wherein the one or more target cells comprise thecells of a subtractive cell line induced to express on their surface atarget epitope, whereby the internalizing antibody moieties that bind tosaid target epitope are selected.
 58. The method of claim 57, whereinthe target cells comprise cells of a subtractive cell line transformedwith a nucleic acid that encodes said target epitope.